Micro-rnas and compositions comprising same for the treatment and diagnosis of serotonin-, adrenalin-, noradrenalin-, glutamate-, and corticotropin-releasing hormone- associated medical conditions

ABSTRACT

microRNAs and compositions comprising same for the treatment and diagnosis of serotonin-, adrenalin-, noradrenalin-, glutamate-, and corticotropin-releasing hormone-associated medical conditions are provided.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.15/873,998 filed on Jan. 18, 2018, which is a division of U.S. patentapplication Ser. No. 14/856,697 filed on Sep. 17, 2015 now U.S. Pat. No.9,901,593, which is a division of U.S. patent application Ser. No.14/236,928 filed on Feb. 4, 2014, now U.S. Pat. No. 9,150,858 which is aNational Phase of PCT Patent Application No. PCT/IB2012/053971 havingInternational Filing Date of Aug. 2, 2012, which claims the benefit ofpriority of U.S. Provisional Patent Application No. 61/514,954 filed onAug. 4, 2011.

The contents of the above applications are all incorporated by referenceas if fully set forth herein in their entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 76317SequenceListing.txt, created on Feb. 12,2019, comprising 33,377 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to microRNAsand, more particularly, but not exclusively, to the use of same fordisease diagnosis, treatment and monitoring treatment.

Mood disorders such as major depression represent some of the mostcommon and proliferating health problems worldwide effecting about 10%of the population. Despite many decades of research, the mechanismsbehind depression onset, susceptibility and available therapies are onlypartially understood. Currently only about a third of patients respondto available treatments, therefore, there is a great need for betterunderstanding of the pathology. The current dogma regarding the etiologyof depression is of a complex interaction between environmental factorsand genetic predisposition, suggesting a mechanistic role for epigeneticprocesses.

Serotonin (5HT) is a monoamine neurotransmitter produced in the brain bythe raphe nucleus (RN), which project extensively throughout the brainto modulate variety of cognitive, emotional and physiological functions.The link between disregulated serotonergic activity and depression iswell established [Michelsen K A. et al., Brain Res Rev (2007)55(2):329-42]. The levels of 5HT, as well as the genetic circuitry incharge of it production, secretion, reuptake and deactivating, aredysregulated in depression. Furthermore, most currently availableantidepressant drugs target the function of 5HT system related proteins,resulting in increased 5HT levels in the synapse [Krishnan V and NestlerE J, Nature (2008) 455: 894-902]. Available therapeutics require a longperiod of administration before relief of symptoms is observed.

MicroRNAs (miRs) are a subset of endogenous small (approximately 22nucleotide) RNA molecules that repress gene expressionpost-transcriptionally. MiRs are transcribed as primary-miR moleculesthat are processed in the cell nucleus into precursor miRs with stemloop structures, which are exported to the cytoplasm where they arefurther processed into the active mature miRs. The mature miR issubsequently incorporated into the RNA-induced silencing complex andfunction primarily by binding to the 3′untranslated regions (3′UTRs) ofspecific mRNA molecules. Binding occurs via the seed sequence, a 6-8nucleotides sequence at the 5′ end of the miR, that base pairs to acomplementary seed match sequence on the target mRNA 3′ UTR. Binding ofa miR leads to direct mRNA destabilization or translational repression,ultimately resulting in reduced protein levels of target gene.

MiRs are abundant in the nervous system, and initial research has mainlyfocused on neurons in the context of development, cancer andneurodegenerative disorders and normal process such as plasticity [KosikK S. Nat Rev Neurosci (2006) 7:911-20]. Additionally, it has beensuggested that miRs play a role in psychiatric disorders such asschizophrenia, autism and also depression and anxiety, both in humansand in mouse models [Miller B H and Wahlestedt C, Brain Res (2010) 1338:89-99]. Several studies have recently demonstrated the involvement ofmiRs in regulating 5HT related genes [Millan M J. Curr Opin Pharmacol(2011) 11(1):11-22] revealing the emerging role of miRs in theregulation of 5HT system and their potential association with depressionrelated disorders.

U.S. Patent Application No. 20100222413 (to Stoffel M. et al.) disclosechemically modified oligonucleotides for modulating expression ofmicroRNAs. U.S. 20100222413 further discloses methods for silencingmicroRNAs (e.g. miR-122, miR-16, miR-192 and miR-194) for the treatmentof diseases of the central nervous system.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a medical condition in which anelevation of serotonin level is therapeutically beneficial in a subjectin need thereof, the method comprising administering to or expressing ina cell of the subject an exogenous polynucleotide encoding at least onemicroRNA or a precursor thereof, wherein the microRNA is selected fromthe group consisting of miR-135, miR-335, miR-26 and miR-182, therebytreating the medical condition.

According to an aspect of some embodiments of the present inventionthere is provided a use of an exogenous polynucleotide encoding at leastone microRNA or a precursor thereof, wherein the microRNA is selectedfrom the group consisting of miR-135, miR-335, miR-26 and miR-182, forthe manufacture of a medicament identified for treating a medicalcondition in which an elevation of serotonin level is therapeuticallybeneficial.

According to an aspect of some embodiments of the present inventionthere is provided a method of increasing a serotonin level in a synapticcleft of a subject in need thereof, the method comprising administeringto or expressing in a serotonergic neuron of the subject an exogenouspolynucleotide encoding at least one microRNA or a precursor thereof,wherein the microRNA is selected from the group consisting of miR-135,miR-335, miR-26 and miR-182, thereby increasing the serotonin level inthe synaptic cleft.

According to an aspect of some embodiments of the present inventionthere is provided an isolated neuroglia cell comprising a nucleic acidconstruct expressing at least one microRNA or a precursor thereof,wherein the microRNA is selected from the group consisting of miR-135,miR-335, miR-26 and miR-182 under a transcriptional control of a cisacting regulatory element.

According to an aspect of some embodiments of the present inventionthere is provided an isolated polynucleotide encoding at least onemicroRNA or a precursor thereof, wherein the microRNA is selected fromthe group consisting of miR-135, miR-335, miR-26 and miR-182, fortreating a medical condition in which an elevation of serotonin level istherapeutically beneficial.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a medical condition in which alow adrenaline or noradrenaline level is therapeutically beneficial in asubject in need thereof, the method comprising administering to orexpressing in a cell of the subject an exogenous polynucleotide encodinga miR-19 or a precursor thereof, thereby treating the medical condition.

According to an aspect of some embodiments of the present inventionthere is provided a use of an exogenous polynucleotide encoding a miR-19or a precursor thereof for the manufacture of a medicament identifiedfor treating a medical condition in which a low adrenaline ornoradrenaline level is therapeutically beneficial.

According to an aspect of some embodiments of the present inventionthere is provided an isolated cell comprising a nucleic acid constructexpressing a miR-19 or a precursor thereof under a transcriptionalcontrol of a cis acting regulatory element.

According to an aspect of some embodiments of the present inventionthere is provided an isolated polynucleotide encoding a miR-19 or aprecursor thereof for treating a medical condition in which a lowadrenaline or noradrenaline level is therapeutically beneficial.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a medical condition in which alow corticotropin-releasing hormone (CRH) level is therapeuticallybeneficial in a subject in need thereof, the method comprisingadministering to or expressing in a cell of the subject an exogenouspolynucleotide encoding a miR-15 or a precursor thereof, therebytreating the medical condition.

According to an aspect of some embodiments of the present inventionthere is provided a use of an exogenous polynucleotide encoding a miR-15or a precursor thereof for the manufacture of a medicament identifiedfor treating a medical condition in which a low corticotropin-releasinghormone (CRH) level is therapeutically beneficial.

According to an aspect of some embodiments of the present inventionthere is provided an isolated cell comprising a nucleic acid constructexpressing a miR-15 or a precursor thereof under a transcriptionalcontrol of a cis acting regulatory element.

According to an aspect of some embodiments of the present inventionthere is provided an isolated polynucleotide encoding a miR-15 or aprecursor thereof for treating a medical condition in which a lowcorticotropin-releasing hormone (CRH) level is therapeuticallybeneficial.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a medical condition in which alow glutamate receptor level is therapeutically beneficial in a subjectin need thereof, the method comprising administering to or expressing ina cell of the subject an exogenous polynucleotide encoding a miR-181 ora precursor thereof, thereby treating the medical condition. Accordingto an aspect of some embodiments of the present invention there isprovided a use of an exogenous polynucleotide encoding a miR-181 or aprecursor thereof for the manufacture of a medicament identified fortreating a medical condition in which a low glutamate receptor level istherapeutically beneficial.

According to an aspect of some embodiments of the present inventionthere is provided an isolated cell comprising a nucleic acid constructexpressing a miR-181 or a precursor thereof under a transcriptionalcontrol of a cis acting regulatory element.

According to an aspect of some embodiments of the present inventionthere is provided an isolated polynucleotide encoding a miR-181 or aprecursor thereof for treating a medical condition in which a lowglutamate receptor level is therapeutically beneficial.

According to an aspect of some embodiments of the present inventionthere is provided a nucleic acid construct comprising a nucleic acidsequence encoding a microRNA or a precursor thereof, wherein themicroRNA or a precursor thereof is selected from the group consisting ofmiR-135, miR-335, miR-26, miR-27, miR-181, miR-182, miR-19 and miR-15,the nucleic acid sequence being under a transcriptional control of a cisacting regulatory element.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising the nucleicacid construct of the present invention and a pharmaceuticallyacceptable carrier or diluent.

According to an aspect of some embodiments of the present inventionthere is provided a method of regulating an expression of a tryptophanhydroxylase 2 (Tph2) gene in a neuroglia cell, the method comprisingmodulating an activity or expression of a microRNA or a precursorthereof in the neuroglia cell, wherein the microRNA is selected from thegroup consisting of miR-181 and miR27, thereby regulating the expressionof the Tph2 gene.

According to an aspect of some embodiments of the present inventionthere is provided a method of regulating an expression of a glutamatereceptor gene in a neuroglia cell, the method comprising modulating anactivity or expression of miR-181 or a precursor thereof in theneuroglia cell, thereby regulating the expression of the glutamatereceptor gene.

According to an aspect of some embodiments of the present inventionthere is provided an isolated polynucleotide comprising a nucleic acidsequence for downregulating an expression of miR-181, miR-27 or aprecursor thereof.

According to an aspect of some embodiments of the present inventionthere is provided a nucleic acid construct comprising a nucleic acidsequence for downregulating an expression of a microRNA or a precursorthereof, wherein the microRNA or a precursor thereof is selected fromthe group consisting of miR-181 and the miR-27, the nucleic acidsequence being under a transcriptional control of a cis actingregulatory element.

According to an aspect of some embodiments of the present inventionthere is provided a method of regulating an expression of a serotonintransporter (Slc6a4) gene in a neuroglia cell, the method comprisingmodulating an activity or expression of a microRNA or a precursorthereof in the neuroglia cell, wherein the microRNA is selected from thegroup consisting of miR-135 and miR-335, thereby regulating theexpression of the Slc6a4 gene.

According to an aspect of some embodiments of the present inventionthere is provided a method of regulating an expression of a serotonininhibitory receptor 1a (Htr1a) gene in a neuroglia cell, the methodcomprising modulating an activity or expression of a microRNA or aprecursor thereof in the neuroglia cell, wherein the microRNA isselected from the group consisting of miR-135, miR-335, miR-181, miR-182and miR-26, thereby regulating the expression of the Htr1a gene.

According to an aspect of some embodiments of the present inventionthere is provided a method of regulating an expression of a DownSyndrome Cell Adhesion Molecule (Dscam) gene in a neuroglia cell, themethod comprising modulating an activity or expression of a miR-182 or aprecursor thereof in the neuroglia cell, thereby regulating theexpression of the Dscam gene.

According to an aspect of some embodiments of the present inventionthere is provided a method of regulating an expression of a Celladhesion molecule L1 (L1cam) gene in a neuroglia cell, the methodcomprising modulating an activity or expression of a miR-182 or aprecursor thereof in the neuroglia cell, thereby regulating theexpression of the L1cam gene.

According to an aspect of some embodiments of the present inventionthere is provided a method of regulating an expression of aTranslin-associated protein X (Tsnax) gene in a neuroglia cell, themethod comprising modulating an activity or expression of a miR-182 or aprecursor thereof in the neuroglia cell, thereby regulating theexpression of the Tsnax gene.

According to an aspect of some embodiments of the present inventionthere is provided a method of regulating an expression of a monoaminehydroxylase (MaoA) gene in a neuroglia cell, the method comprisingmodulating an activity or expression of a miR-27, thereby regulating theexpression of the MaoA gene.

According to an aspect of some embodiments of the present inventionthere is provided a method of regulating an expression of a betaadrenergic receptor 1 (Adrb1) gene in a neuroglia cell or cardiac cell,the method comprising modulating an activity or expression of a miR-19,thereby regulating the expression of the Adrb1 gene.

According to an aspect of some embodiments of the present inventionthere is provided a method of regulating an expression of a canabinoidreceptor 1 (CB1) gene in a neuroglia cell, the method comprisingmodulating an activity or expression of a miR-19 or a precursor thereofin the neuroglia cell, thereby regulating the expression of the CB1gene.

According to an aspect of some embodiments of the present inventionthere is provided a method of regulating an expression of a CRH type 1receptor gene in a neuroglia cell, the method comprising modulating anactivity or expression of a miR-15, thereby regulating the expression ofthe CRH type 1 receptor gene.

According to an aspect of some embodiments of the present inventionthere is provided a method of regulating an expression of a FK506binding protein 5 (FKBP5) gene in a neuroglia cell, the methodcomprising modulating an activity or expression of a miR-15 or aprecursor thereof in the neuroglia cell, thereby regulating theexpression of the FKBP5 gene.

According to an aspect of some embodiments of the present inventionthere is provided a method of regulating an expression of a syntaxin 1a(Stx1a) gene in a neuroglia cell, the method comprising modulating anactivity or expression of a miR-15 or a precursor thereof in theneuroglia cell, thereby regulating the expression of the Stx1a gene.

According to an aspect of some embodiments of the present inventionthere is provided a method of regulating an expression of aserum/glucocorticoid_regulated kinase (Sgk1) gene in a neuroglia cell,the method comprising modulating an activity or expression of a miR-15or a precursor thereof in the neuroglia cell, thereby regulating theexpression of the Sgk1 gene.

According to an aspect of some embodiments of the present inventionthere is provided a method of regulating an expression of a beta 2adrenergic receptor (Adrb2) gene in a neuroglia cell, the methodcomprising modulating an activity or expression of a miR-15 or aprecursor thereof in the neuroglia cell, thereby regulating theexpression of the Adrb2 gene.

According to an aspect of some embodiments of the present inventionthere is provided a method of monitoring treatment of an anti-depressantdrug, the method comprising: (a) treating a subject in need thereof withan anti-depressant drug; and (b) measuring an expression level of amiR-135 in the blood of the subject prior to and following thetreatment, wherein a lower expression level of the miR-135 following tothe treatment by the anti-depressant drug as compared to the expressionlevel of the miR-135 prior to the treatment by the anti-depressant drugis indicative of the efficient treatment.

According to an aspect of some embodiments of the present inventionthere is provided a method of diagnosing a serotonin-related medicalcondition in a subject in need thereof, the method comprising measuringan expression level of a miR-135 in a blood of the subject, wherein ahigh expression level of the miR-135 as compared to that in a bloodsample of a healthy subject is indicative of the serotonin-associatedmedical condition.

According to some embodiments of the invention, the cell is a neurogliacell.

According to some embodiments of the invention, the neuroglia cell is aserotonergic neuron.

According to some embodiments of the invention, the miR-135 is as setforth in SEQ ID NO: 58-62.

According to some embodiments of the invention, the miR-335 is as setforth in SEQ ID NO: 63-64.

According to some embodiments of the invention, the miR-26 is as setforth in SEQ ID NO: 65-69.

According to some embodiments of the invention, the miR-182 is as setforth in SEQ ID NO: 70-71.

According to some embodiments of the invention, the medical condition isselected from the group consisting of a depression, an anxiety, astress, a fatigue, an impaired cognitive function, a panic attack, acompulsive behavior, an addiction, a social phobia, a sleep disorder, afood related disorder, a growth disorder and a reproduction disorder.

According to some embodiments of the invention, when the microRNA ismiR-135, the medical condition is depression or anxiety.

According to some embodiments of the invention, the cell is a neurogliacell or a cardiac cell.

According to some embodiments of the invention, the miR-19 is as setforth in SEQ ID NO: 72-76.

According to some embodiments of the invention, the medical condition isselected from the group consisting of a stress, an anxiety, a memoryimpairment and a heart condition.

According to some embodiments of the invention, the miR-15 is as setforth in SEQ ID NO: 77-80.

According to some embodiments of the invention, the medical condition isselected from the group consisting of a depression, an anxiety, astress, a fatigue, an impaired cognitive function, a panic attack, acompulsive behavior, an addiction, a social phobia, a sleep disorder, afood related disorder, a growth disorder and a reproduction disorder.

According to some embodiments of the invention, the polynucleotide beingunder a transcriptional control of a cis acting regulatory elementactive in a neuroglia cell.

According to some embodiments of the invention, the polynucleotide beingunder a transcriptional control of a cis acting regulatory elementactive in a cardiac cell.

According to some embodiments of the invention, the miR-181 is as setforth in SEQ ID NO: 85-94.

According to some embodiments of the invention, the medical condition isselected from the group consisting of seizures, Huntington's disease,Schizophrenia, Fragile X syndrome, generalized anxiety disorder andcancer.

According to some embodiments of the invention, the cis actingregulatory element is active in a neuroglia cell or a cardiac cell.

According to some embodiments of the invention, the subject is a humansubject.

According to some embodiments of the invention, when the regulatingcomprises upregulating the expression of the Tph2 gene, the modulatingcomprises downregulating the miR-181 and/or the miR-27 in the neurogliacell.

According to some embodiments of the invention, the method furthercomprises measuring an expression of the Tph2 gene following thedownregulating of the miR-181 and/or the miR-27 in the neuroglia cell.

According to some embodiments of the invention, the glutamate receptorgene is selected from the group consisting of glutamate receptormetabotropic 1 (Grm1), glutamate receptor ionotropic, kainate 3 (Grik3),glutamate receptor metabotropic 5 (Grm5), glutamate receptor ionotropickainate 2 (Grik2) and glutamate receptor metabotropic 7 (Grm7).

According to some embodiments of the invention, when the regulatingcomprises downregulating the expression of the Slc6a4 gene, themodulating comprises upregulating the miR-135 and/or miR-335 in theneuroglia cell.

According to some embodiments of the invention, the method furthercomprises measuring an expression of the Slc6a4 gene following theupregulating the miR-135 and/or miR-335 in the neuroglia cell.

According to some embodiments of the invention, when the regulatingcomprises downregulating the expression of the Htr1a gene, themodulating comprises upregulating the miR-135, miR-335, miR-181, miR-182and/or miR-26 in the neuroglia cell. According to some embodiments ofthe invention, the method further comprises measuring an expression ofthe Htr1a gene following the upregulating the miR-135, miR-335, miR-181,miR-182 and/or miR-26 in the neuroglia cell.

According to some embodiments of the invention, when the regulatingcomprises downregulating the expression of the MaoA gene, the modulatingcomprises upregulating the miR-27 in the neuroglia cell.

According to some embodiments of the invention, the method furthercomprises measuring an expression of the MaoA gene following theupregulating the upregulating the miR-27 in the neuroglia cell.

According to some embodiments of the invention, when the regulatingcomprises downregulating the expression of the Adrb1 gene, themodulating comprises upregulating the miR-19 in the neuroglia cell orthe cardiac cell.

According to some embodiments of the invention, the method furthercomprises measuring an expression of the Adrb1 gene following theupregulating the miR-19 in the neuroglia cell or the cardiac cell.

According to some embodiments of the invention, when the regulatingcomprises downregulating the expression of the CB1 gene, the modulatingcomprises upregulating the miR-19 in the neuroglia cell.

According to some embodiments of the invention, the method furthercomprises measuring an expression of the CB1 gene following theupregulating the CB1 in the neuroglia cell.

According to some embodiments of the invention, when the regulatingcomprises downregulating the expression of the CRH type 1 receptor gene,the modulating comprises upregulating the miR-15 in the neuroglia cell.

According to some embodiments of the invention, the method furthercomprises measuring an expression of the CRH type 1 receptor genefollowing the upregulating the miR-15 in the neuroglia cell.

According to some embodiments of the invention, when the regulatingcomprises downregulating the expression of the FKBP5 gene, themodulating comprises upregulating the miR-15 in the neuroglia cell.

According to some embodiments of the invention, the method furthercomprises measuring an expression of the FKBP5 gene following theupregulating the miR-15 in the neuroglia cell.

According to some embodiments of the invention, the method furthercomprises obtaining a blood sample from the subject prior to thetreatment.

According to some embodiments of the invention, the anti-depressant drugis selected from the group consisting of selective serotonin reuptakeinhibitors (SSRI), tricyclic antidepressants and noradrenaline reuptakeinhibitors (NRI).

According to some embodiments of the invention, the serotonin-associatedmedical condition is a psychiatric condition.

According to some embodiments of the invention, the psychiatriccondition is selected from the group consisting of a depression, ananxiety, a stress, a fatigue, an impaired cognitive function, a panicattack, a compulsive behavior, an addiction, a social phobia, a sleepdisorder and a food related disorder.

According to some embodiments of the invention, the miR-135 comprisesmiR-135a or miR-135b.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-1I depict microRNA expression in serotonin (5HT) neurons. FIG.1A is a graphic illustration of differentially expressed miRNAs in 5HTneurons. Lowess normalized values are depicted as 1n2 fold change ofspot intensity plotted against average log intensities (MA plot); FIG.1B is a validation of array results in miRs real time PCR indicatingincreased levels of miR-375 in the 5HT neurons compared to control. n=55HT cells, n=4 non 5HT. Bars represent mean±s.e.m. **P=0.0071; FIG. 1Cis a validation of array results in miRs real time PCR indicatingdecreased levels of miR-135a in the 5HT neurons compared to control. N=55HT cells, n=4 non 5HT. **P=0.0075; FIG. 1D is a van diagramrepresenting crossing bioinformatics predictions for Slc6a4 with 5HTmicroarray results and listing miRs chosen for in vitro testing; FIG. 1Eis a van diagram representing crossing bioinformatics predictions forHtr1a with 5HT microarray results and listing miRs chosen for in vitrotesting; FIG. 1F is a van diagram representing crossing bioinformaticspredictions for Tph2 with 5HT microarray results and listing miRs chosenfor in vitro testing; FIG. 1G is a van diagram representing crossingbioinformatics predictions for MaoA with 5HT microarray results andlisting miRs chosen for in vitro testing; FIG. 1H is a graphillustrating luciferase reporter assay results indicating that miR-181cand miR-27b may target Tph2 3′UTR; and FIG. 1I is a graph illustratingluciferase reporter assay results indicating that miR-27b may targetHtr1a MaoA.

FIGS. 2A-2H depict microRNA targeting of Slc6a4 3′UTR (SEQ ID NO: 25)and Htr1a 3′UTR (SEQ ID NO: 27). FIG. 2A is an illustration of miR-135aand miR-135b (SEQ ID NOs: 24 and 26, respectively) targeting of Slc6a43′UTR; FIG. 2B is an illustration of miR-135a and miR-135b (SEQ ID NOs:24 and 26, respectively) targeting of Htr1a 3′UTR; FIG. 2C is a graphillustrating luciferase reporter assay results indicating that miR-135aand miR-135b may target Slc6a4 3′UTR. Luciferase assay data depictsrenilla luciferase activity normalized to the activity of aco-transfected firefly luciferase reporter in HEK293 cells transfectedwith 3′UTR of the gene described and an empty vector, or a vectorover-expressing a specific miR. Bars represent mean±s.e.m. *P=0.014,***P=0.0002, for miR-16 #p<0.0535, for miR-27 #P=0.0967; FIG. 2D is agraph illustrating luciferase reporter assay results indicating thatmiR-135a, miR-135b, miR-335, miR-181C and miR-26a may target Htr1a3′UTR. ***P<0.0001, **P=0.0029; FIG. 2E is an illustration of slc6a43′UTR conservation of the seed matches for miR-135 (SEQ ID NOs: 27-41);FIG. 2F is an illustration of Htr1a 3′UTR seed matches for miR-135 (SEQID NOs: 42-54), indicating seed 1 appearing only in mouse 3′ UTR, andseed 2 is highly conserved; FIG. 2G is a graph illustrating thatmutation in miR-135 seed match in slc6a4 3′UTR blocked the repressoreffect of miR-135a and miR-135b. ***P<0.0001, **P=0.0032; and FIG. 2H isa graph illustrating mutation in miR-135 seed matches in Htr1a 3′ UTRindividually and both together, indicating miR-135b targets Htr1a viaboth the seed matches and miR-135a only by seed 2. ***P<0.0001.

FIGS. 3A-3J depict miR-135a and miR-135b levels under differentconditions.

FIG. 3A is a graph illustrating down-regulation of miR-135a levels inthe RN following acute stress. Bars represent mean±s.e.m. (n=8 in the 0group, n=10 in the 90 group and n=9 in the 24 group) ***P<0.0001,*P=0.0357; FIG. 3B is a graph illustrating down-regulation of miR-135blevels in the RN following acute stress. ***P<0.0001, **P=0.0055; FIG.3C is a graph illustrating up-regulation of miR-135a levels in the RNfollowing acute and chronic imipramine administration independently fromwhether the mice were exposed to social defeat. (n=8 in control chronicsaline and control chronic imipramine, n=7 acute imipramine, n=11 socialdefeat chronic saline, n=9 in the social defeat chronic imipramine)**P=0.003; FIG. 3D is a graph illustrating up-regulation of miR-135blevels in the RN following acute and chronic imipramine administrationindependently from whether the mice were exposed to social defeat.**P=0.0093; FIG. 3E is a graph illustrating increase in miR-135a levelsin the RN following acute or chronically administrated SSRI, and not NRIor saline. (n=8 in each group apart from acute saline n=7) ***P<0.0001;FIG. 3F is a graph illustrating an unaltered miR-135b levels in the RNfollowing acute or chronically administered SSRI or NRI; FIG. 3G is agraph illustrating decrease in miR-135a levels in the plasma of micereceiving chronic or acute SSRI as compared to controls. (n=8 in eachgroup apart from chronic SSRI and NRI n=7) **P=0.0004 for acute SSRIcompared to acute saline and **P=0.0006 for the chronic SSRI compared tothe chronic saline; FIG. 3H is a graph illustrating unchanged miR-135blevels in the plasma of mice receiving chronic or acute SSRI as comparedto controls; FIG. 3I is a scatter plot graph demonstrating individualmice miR-135a levels in the RN compared to the plasma indicating areverse correlation in mice receiving SSRI or saline treatment; and FIG.3J is a scatter plot graph demonstrating no correlation between miR-135blevels in the RN to the plasma in mice receiving SSRI treatment comparedto controls.

FIGS. 4A-4H depict in vivo over-expression of miR-135b. FIG. 4A is aschematic illustration of lentiviruses for over-expression of miR-135b;FIG. 4B is a graph illustrating real time PCR results indicatingover-expression of miR-135b in vivo in the dorsal raphae nucleus (DRN)of adult mice. Bars represent mean±s.e.m. (n=5 GFP injected and n=3miR-135 OE) P=0.0032; FIGS. 3C-3D are illustrations of a DRN injectionsite by demonstration of GFP staining at injections site. (Section mapadopted from Paxinos); FIG. 4E is a graph illustrating decreasedimmobility time in the forest swim test in mice over-expressing miR-135bin the RN compared to control mice. (n=9 control n=9 miR-135) P=0.0088in minute 3 and P=0.00330 for minute 4;

FIG. 4F is a graph illustrating decreased immobility time in the tailsuspension test in mice over-expressing miR-135b in the RN compared tocontrol mice. P=0.07351; FIGS. 4G-4H are graphs illustrating nodifference in home cage locomotion in of mice over-expressing miR-135bin the RN compared to controls.

FIG. 5 depicts ADRb1 3′UTR cloned following the luciferase gene.Illustration of intact (top) ADRb1 3′UTR, harboring four miR-19 bindingsites, and mutant (bottom) form of ADRb1 3′UTR, lacking all four miR-19binding sites, cloned downstream to the luciferase gene in Psicheck2plasmid.

FIGS. 6A-6E depict that miR-19b targets ADRb1 3′UTR via seed matches onits 3′UTR; FIGS. 6A-6B are graphs illustrating normalized luciferaselevels measured in HT22 cells that express low endogenous miR-19 levelsfollowing transfection with (FIG. 6A) GFP plasmid or (FIG. 6B)pre-miR-19b overexpression (OE) plasmid; FIGS. 6C-6E are graphsillustrating normalized luciferase levels measured in HEK293T cells thatexpress high endogenous miR-19 levels. Transfection with (FIG. 6C)control plasmid, (FIG. 6D) miR-19b knockdown (KD) probe or scrambledprobe as control, and (FIG. 6E) transfection with miR-19b miArrestplasmid or control miArrest plasmid. *** P<0.005. Renilla luciferaseactivity was normalized by firefly luciferase expression levels andpresented as ratio of activity achieved by the mutant form ofAdrb1-3′UTR (Adrb1-mut) at the presence of control treatment.

FIGS. 7A-7D depict differential expression of miRNA in the amygdale.FIGS. 7A-7B are graphs illustrating differential expression of miRNA inthe amygdala 90 minutes following acute stress. FIG. 7A illustratesagilent array results. FIG. 7B illustrates affymetrix array results.Normalized values are depicted as log 2 ratio (stress vs. control) ofspot intensity plotted against average intensities across conditions(N=2,2). The intensity of each miRNA was calculated as the averagenormalized intensity across biological repeats. miR-15a and miR-15b areindicated in red. miR-124, a well-established neuronal marker notaffected by the stress protocol is indicated in white; FIG. 7Cillustrates that miR-15a and miR-15b have a semi-conserved seed match oncorticotropin releasing hormone type 1 receptor 3′UTR [CRHR1, adaptedfrom targetscan(dot)org]; and FIG. 7D is a graph illustrating luciferaseactivity measured in HEK293T cells co-transfected with miR-15b-EGFPover-expressing or GFP expressing plasmid and a luciferase reporterplasmid controlled by CRFR1-3′UTR. Renilla luciferase activity wasnormalized by firefly luciferase expression levels.

FIG. 8 is a graph illustrating luciferase reporter assay resultsindicating that miR-182 probably targets Htr1a 3′UTR. Luciferase assaysdata depicts renilla luciferase activity normalized to the activity of aco-transfected firefly luciferase reporter in HEK293 cells transfectedwith 3′UTR of the gene described and an empty vector, or a vectorover-expressing a specific miR.

FIG. 9 is a graph illustrating real time PCR results of miR-182expression levels in adult mice DRN indicating a trend for decreasedexpression following chronic social defeat. Data represents mean±SEM n=7controls and 18 mice in social defeat group, #=p=0.1.

FIG. 10 is a van diagram representing in silico bioinformaticspredictions for miR-182 targets in two algorithms, and list of potentialtarget genes highly relevant for normal and pathological neuronalfunction appearing in this prediction.

FIGS. 11A-11C depict over-expression or knockdown of miR-182. FIG. 11Ais a schematic illustration of lentiviruses for over-expression ofmiR-182; FIG. 11B is a graph illustrating real time PCR resultsindicating over-expression of miR-182 in vitro in N2A cell line; andFIG. 11C is a schematic illustration of lentiviruses for knockdown ofmiR-182.

FIGS. 12A-12D depict miR-19 levels in the PFC following NRIadministration. The NRI reboxetine was administrated either acutely(once) or chronically (for 18 days). Of note, miR-19a and miR-19b levelsdecreased in the PFC following acute administration of NRI (FIG. 12A andFIG. 12B, respectively) but increased following chronic administrationof NRI (FIG. 12C and FIG. 12D, respectively).

FIGS. 13A-13D depict miR-19 levels in the PFC and amygdala of micesubjected to social defeat. miR-19a and miR-19b levels were measured insamples from amygdala taken from mice that were subjected to socialdefeat paradigm. Of note, miR-19a and miR-19b levels in the PFC wereelevated in mice categorized as being “Susceptible” to social defeatrelative to control mice (FIG. 13A and FIG. 13B, respectively). miR-19levels were also elevated in the amygdala of mice categorized as being“Susceptible” to social defeat relative to control mice (FIG. 13C andFIG. 13D, respectively).

FIG. 14 depicts miRNA-19b targeting CB1 3′UTR. Transfection of HT-22cells with CB1 3′ UTR and plasmids overexpressing either miR-19b or GFPcontrol lead to a 50% decrease in normalized luciferase levels.

FIGS. 15A-15B are schematic illustrations of a coronal section of themouse brain. FIG. 15A shows several nuclei in the brain including theBLA (adapted from the mouse brain by Paxinos and Franklin); FIG. 15Bshows a CB1 distribution in the brain (adapted from Allen Brain Atlaswww(dot)mouse(dot)brain-map(dot)org/). Of note, it is evident by thisdistribution that CB1 is abundant in the BLA.

FIG. 16 is a schematic illustration of a proposed mechanism for memoryconsolidation in the basolateral nucleus of the amygdala (BLA).Corticosterone (CORT) binds to a yet-uncharacterized membrane-boundglucocorticoid receptor (mbGR) that activates the Gs-cAMP/PKA pathway toinduce endocannabinoid (eCB) synthesis. Endocannabinoids are releasedinto the synapse where they bind to CB1 receptors on GABAergic terminalsinhibiting GABA release. This inhibition of GABA release disinhibitsnorepinephrine (NE) release and increases NE activation of postsynapticβ-adrenoreceptors, increasing the consolidation of emotionally-aversivememories.

FIGS. 17A-17B illustrate Ago2 in the RISC complex. FIG. 17A is aschematic illustration of Ago2 in the RISC complex, mediating theinteraction between the miRNA and the mRNA; FIG. 17B illustrates awestern blot analysis performed with anti-Ago2 antibody. This IP wasspecific to the Ago2 protein as can be seen when comparing the totalbrain sample that was precipitated once with the Ago2 antibody and oncewith the IgG1 control. Of note, there was no detection of the Ago2protein on the samples precipitated with the IgG1 control.

FIGS. 18A-18D depict a social avoidance test. Mice were placed in a mazefor 3 minutes alone for habituation (FIG. 18A and FIG. 18B) and theirmovement was recorded and plotted. After 3 minutes a novel ICR mouse wasplaced in the chamber next to the examined mouse (FIG. 18C and FIG. 18D)and the movement of the examined mouse was recorded and plotted again.

FIG. 19A depicts a heatmap illustration of selected miRNAs up regulatedin the arrays.

FIG. 19B depicts a heatmap illustration of selected miRNAs downregulated in the arrays.

FIGS. 20A-20B depict a log 2 expression of miR-15a (FIG. 20A) and FKBP5(FIG. 20B) from the microarray results. Each red dot refers to onerepetition of an array. The control group (CNT) had 4 repetitions, the“Susceptible” group (SUSC) had 3 repetitions and the “Resilient” group(RESIL) had 3 repetitions. The black line showed the mean of therepetitions in each group.

FIG. 20C depicts a 3′ UTR sequence of mouse FKBP5 (taken fromtargetscan(dot)org).

FIGS. 21A-21B depict the levels of amygdalar miR-15a (FIG. 21A) andFKBP5 (FIG. 21B) in “Susceptible” mice relative to control micefollowing social defeat. Of note, miR-15a levels were elevated in theamygdala of mice subjected to social defeat and characterized as“Susceptible” (FIG. 21A). FKBP5 levels were decreased in the amygdala ofmice subjected to social defeat and characterized as “Susceptible” (FIG.21B).

FIG. 22 is a schematic illustration of the 3′UTR of Stx1a, Sgk1 andAdrb2, each harboring a single miRNA-15 binding site.

FIG. 23 depicts the levels of amygdalar miR-181 in mice subjected tosocial defeat relative to control mice. Of note, miR-181 levels wereelevated in the amygdala of mice subjected to social defeat.

FIG. 24 depicts Van diagrams representing crossing bioinformaticspredictions for miR-181 and glutamate receptors.

FIG. 25 is a schematic illustration of intact 3′UTR of 6 potentialtargets of miR-181.

FIG. 26 depicts expression levels of miR182 in the raphe nucleusfollowing stress. Of note, an acute 30 minute immobilization stress ledto decreased expression levels of miR182 in the RN when tested 24 hoursfollowing the stress as measured by real time PCR. **=P<0.01; n=8 ineach group.

FIGS. 27A-27C depict results of a luciferase reporter assay indicatingthat miR182 targets DSCAM, L1CAM and TSNAX 3′UTR. FIG. 27A illustratesdata of luciferase assays depicting renila luciferase activitynormalized to the activity of a co-transfected firefly luciferasereporter in N2a cells transfected with 3′UTR of the genes described andan empty vector, or a vector over-expressing a specific miR. Mutation inmiR182 seed match in L1cam (FIG. 27B) and Tsnax (FIG. 27C) 3′ UTRsblocked the represoric effect of miR182. Bars represent mean±s.e.m.*P<0.05, **P<0.01, ***P<0.001.

FIGS. 28A-28D depict expression of miR135 in the amygdala (AMY) and theprefrontal cortex (PFC). FIG. 28A illustrates that acute SSRI and NRIincreased miR135a levels in the AMY; FIG. 28B illustrates that miR135blevels in the AMY were upregulated by acute SSRI or NRI administrationcompared to saline; FIG. 28C illustrates that chronic SSRI decreasedmiR135a levels in the PFC; and FIG. 28D illustrates that miR135b levelsin the PFC were upregulated by acute SSRI or NRI and decreased bychronic SSRI treatment. n=7-8 in each group*=P<0.05;**=P<0.01;***=P<0.0001.

FIGS. 29A-29B depict increased miR135 levels in mice circulation systemfollowing social defeat. miR135a (FIG. 29A) and miR135b (FIG. 29B)levels in plasma of mice two weeks following social defeat weresignificantly increased compared to control mice (**=P<0.01 n=7-16 ineach group).

FIGS. 30A-30E depict validation of miR135 KD in vitro and in vivo. FIGS.30A-30B illustrate results of a luciferase reporter assay indicatingmiR135 targeting of Htr1a (FIG. 30A) and slc6a4 (FIG. 30B) was blockedby the miR135b KD construct; FIG. 30C is a schematic illustration ofmiR135bKD and control viral vectors; and FIGS. 30D-30E are illustrationsof a DRN injection site (FIG. 30D adopted from Paxinos), and FIG. 30E isa GFP staining of DRN infected with miR135 KD lentiviruses.

FIGS. 31A-31G depict increased anxiety-like behavior and attenuatedresponse to SSRI in miR135KD mice. FIG. 31A illustrates that thebehavior of miR135KD mice was similar to control mice in the open fieldtest; FIG. 31B illustrates increased anxiety-like behavior in miR135KDmice compared to control mice in the elevated pulse maze; FIG. 31Cillustrates that in the dark light transfer test miR135KD mice spentmore time in the light chamber compared to control mice under basalstress conditions, but not following acute stress; FIG. 31D illustratesthat miR135KD mice visited the light chamber more times compared tocontrol mice, under basal stress conditions, but not following acutestress; FIG. 31E illustrates that miR135KD mice traveled less distancein the light chamber compared to control mice, under basal stressconditions, but not following acute stress; FIG. 31F illustrates nodifference between miR135KD mice and control mice in tail suspensiontest both in basal conditions and following SSRI administration, yetreduction in immobility time was observed following SSRI treatmentcompared to basal condition in both groups (FIGS. 31F-31G). Immobilitytime was reduced by SSRI in both groups, however the reduction wasattenuated in miR135KD mice compared to controls in the last 2 minutesof the test.˜=p<0.1 *=p<0.05;**=p<0.01; ***=p<0.001. n=10-11 in eachgroup.

FIG. 32 is a schematic illustration of miR135 mice inducibleoverexpression system. Transgenic mice expressing floxed transactionalstop before miR135a sequence and GFP reporter. Mutant transgenic miceexpress miR135a only in 5-HT ePet positive cells.

FIGS. 33A-33C depict validation of a mice line overexpressing miR135 in5-HT neurons. FIG. 33A illustrates that miR135 was overexpressed in theRN of miR135OE mice compared to control mice. FIGS. 33B-33C illustratethat miR 135 target genes mRNA were downregulated in miR135OE mice RN,both Slc6a4 (FIG. 33B) and Htr1a (FIG. 33C). #=p<0.1 *=p<0.05; n=4 ineach group.

FIGS. 34A-34E depict decreased anxiety and depression-like behaviorfollowing social defeat in miR135OE mice. FIG. 34A shows that miR135OEmice have a decreased anxiety-like behavior in the open field test; FIG.34B shows less anxiety like behaviors compared to control of miR135OEmice in a dark light transfer test;

FIG. 34C shows decreased anxiety-like behavior compared to control inelevated pulse maze of miR135OE mice; FIG. 34D shows tendency towardsdecreased immobility time of miR135OE mice compared to controls in tailsuspension test; and FIG. 34E shows reduced immobility time in miR135OEmice compared to controls in the forced swim test.#=p<0.1*=p<0.05;**=p<0.01 n=7-11 in each group.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to microRNAsand, more particularly, but not exclusively, to the use of same fordisease, diagnosis, monitoring and treatment.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways. Also,it is to be understood that the phraseology and terminology employedherein is for the purpose of description and should not be regarded aslimiting.

The link between dysregulated serotonergic activity and psychiatricdisorders such as anxiety and depression has been previouslyestablished, yet the molecular mechanisms underlying these pathologiesare not fully understood. MicroRNAs (miRs) are a subset of small RNAmolecules that regulate gene expression post-transcriptionally and areabundant in the brain.

While reducing the present invention to practice, the present inventorshave uncovered that specific microRNAs (miRs) are involved in regulationof serotonin (5HT) neuro-glia related genes and are thus involved inmodulating medical conditions associated with aberrant serotonin levelssuch as psychiatric disorders.

As is illustrated hereinbelow and in the Examples section which follows,the present inventors determined the miRs expression pattern in 5HTneurons, obtained from the raphe nucleus (RN) of 5HT reporter mice(ePET-YFP), using miRs microarray (see Tables 2A-B in the Examplessection which follows). The unique miRs expression profile ofserotonergic neurons obtained from the array was bioinformaticallyanalyzed to indentify miRs that putatively target key serotonergicrelated genes, such as serotonin transporter (Slc6a4, FIG. 1D),serotonin auto receptor (Htr1a, FIG. 1E), tryptophan hydroxylase 2(Tph2, FIG. 1F) and monoamine hydroxylase (MaoA, FIG. 1G). miRNAtargeting of the 3′UTRs for these genes were further tested in vitroillustrating specific miRs (e.g. miR-135) that specifically target andregulate the 5HT neuronal genes (see FIGS. 1H-1I and FIGS. 2C-2D). Thepresent inventors have further illustrated that miR-135 expressionlevels are altered in the RN and plasma following acute stress (FIGS.3A-3D) and following treatment with antidepressants (FIGS. 3E-3J). Invivo miR-135 over-expression in the RN of adult mice reduceddepression-like behaviors following social defeat (FIGS. 4A-4H).Moreover, the present inventors have illustrated the activity of miR-182as a regulator of neuronal activity (via direct repression of Htr1a,FIG. 8) and of psychopathological behavior (FIG. 9) and of miR-15 asregulator of stress response [via direct repression of CRH1R (FIGS.7A-7B), FK506 binding protein 5 (FKBP5) (FIGS. 21A-21B) and Stx1a, Sgk1and Adrb2 (FIG. 22)]. The present inventors have also illustrated thespecific targeting of beta adrenergic receptor (Adrb1) and canabinoidreceptor 1 (CB1) by miR-19. miR-19 over-expression repressed Adrb1(FIGS. 6A-6C) while knockdown of miR-19 enhanced Adrb1 expression (FIGS.6D-6E). miR-19 over-expression also repressed CB1 (FIG. 14). The presentinventors have also uncovered targets for miR-181. Specifically, thepresent inventors have illustrated that miR-181 specifically regulatesglutamate receptors (FIGS. 24 and 25). Taken together, these resultssubstantiate the use of miRNAs or sequences regulating same, such asmiR-135, miR-335, miR-181, miR-182, miR-26, miR-27, miR-15 and miR-19,as therapeutic modalities.

Thus, according to one aspect of the present invention there is provideda method of treating a medical condition in which an elevation ofserotonin level is therapeutically beneficial in a subject in needthereof, the method comprising administering to or expressing in a cellof the subject an exogenous polynucleotide encoding at least onemicroRNA or a precursor thereof.

According to a specific embodiment, for treating a medical condition inwhich an elevation of serotonin level is therapeutically beneficial, themicroRNA comprises miR-135, miR-335, miR-26 and miR-182.

According to another aspect of the present invention there is provided amethod of treating a medical condition in which a low adrenaline ornoradrenaline level is therapeutically beneficial in a subject in needthereof, the method comprising administering to or expressing in a cellof the subject an exogenous polynucleotide encoding a microRNA or aprecursor thereof.

According to a specific embodiment, for treating a medical condition inwhich a low adrenaline or noradrenaline level is therapeuticallybeneficial, the microRNA comprises miR-19.

According to another aspect of the present invention there is provided amethod of treating a medical condition in which a lowcorticotropin-releasing hormone (CRH) level is therapeuticallybeneficial in a subject in need thereof, the method comprisingadministering to or expressing in a cell of the subject an exogenouspolynucleotide encoding a microRNA or a precursor thereof.

According to a specific embodiment, for treating a medical condition inwhich a low corticotropin-releasing hormone (CRH) level istherapeutically beneficial, the microRNA comprises miR-15.

According to another aspect of the present invention there is provided amethod of treating a medical condition in which a low glutamate receptorlevel is therapeutically beneficial in a subject in need thereof, themethod comprising administering to or expressing in a cell of thesubject an exogenous polynucleotide encoding a microRNA or a precursorthereof.

According to a specific embodiment, for treating a medical condition inwhich a low glutamate receptor level is therapeutically beneficial, themicroRNA comprises miR-181.

The term “treating” refers to inhibiting or arresting the development ofa disease, disorder or condition and/or causing the reduction,remission, or regression of a disease, disorder or condition or keepinga disease, disorder or medical condition from occurring in a subject whomay be at risk for the disease disorder or condition, but has not yetbeen diagnosed as having the disease disorder or condition. Those ofskill in the art will understand that various methodologies and assayscan be used to assess the development of a disease, disorder orcondition, and similarly, various methodologies and assays may be usedto assess the reduction, remission or regression of a disease, disorderor condition.

As used herein, the term “subject” includes mammals, preferably humanbeings at any age which suffer from the pathology. Preferably, this termencompasses individuals who are at risk to develop the pathology.

As used herein the phrase “medical condition in which an elevation ofserotonin level is therapeutically beneficial” refers to a disease ordisorder in which increasing the level of serotonin can prevent anoccurrence of a disease or medical symptoms associated therewith or haltdisease progression or medical symptoms associated therewith (as furtherdetailed hereinbelow).

As used herein, the term “serotonin” refers to the monoamineneurotransmitter [also referred to as 5-hydroxytryptamine (5-HT)].Serotonin is set forth e.g. in CAS number 50-67-9.

According to one embodiment, there is provided a method of increasing aserotonin level in a synaptic cleft, the method comprising administeringto or expressing in a neuroglia cell e.g., serotonergic neuron of thesubject an exogenous polynucleotide encoding at least one microRNA or aprecursor thereof.

As used herein, the term “synaptic cleft” refers to the area between twoneurons through which electrical or chemical signals pass.

A “neuroglia cell” refers to a neuron or a glial cell (e.g.,oligodendrocytes or astrocyte).

As used herein, the term “serotonergic neuron” refers to a neuron whichsecretes serotonin or is capable of serotonin reuptake (i.e. byserotonin transporters expressed on their cell surfaces).

The medical condition in which an elevation of serotonin level istherapeutically beneficial may comprise, for example, any mood disorderincluding depression, anxiety, stress, fatigue, impaired cognitivefunction, panic attack, compulsive behavior, addiction, social phobia;sleep disorder, food related disorder, growth disorder and reproductiondisorder.

According to a specific embodiment, the medical condition in which anelevation of serotonin level is therapeutically beneficial comprisesdepression.

According to one embodiment, when the medical condition is depression oranxiety, the microRNA is miR-135.

It will be appreciated that the depression or anxiety may notnecessarily be related to serotonin.

As used herein the phrase “medical condition in which a low adrenalineor noradrenaline level is therapeutically beneficial” refers to adisease or disorder in which decreasing the expression or activity ofadrenaline or noradrenaline can prevent an occurrence of a disease ormedical symptoms associated therewith or halt disease progression ormedical symptoms associated therewith (as further detailed hereinbelow).

As used herein, the term “adrenaline” refers to the hormone andneurotransmitter (also known as epinephrine). Adrenaline is set forthe.g. in CAS number 51-43-4.

As used herein, the term “noradrenaline” refers to the catecholamineacting as a hormone and neurotransmitter (also known as norepinephrine).Noradrenaline is set forth e.g. in CAS numbers (1) 51-41-2 (1) and138-65-8(d1).

The medical condition in which a low adrenaline or noradrenaline levelis therapeutically beneficial may comprise, for example, stress-relateddisorder, anxiety, memory impairment, heart conditions (e.g.palpitations, tachycardia and arrhythmia), headaches, tremors,hypertension, and acute pulmonary edema.

As used herein the phrase “medical condition in which a lowcorticotropin-releasing hormone (CRH) level is therapeuticallybeneficial” refers to a disease or disorder in which decreasing theexpression or activity of CRH can prevent an occurrence of a disease ormedical symptoms associated therewith or halt disease progression ormedical symptoms associated therewith (as further detailed hereinbelow).

As used herein, the term “corticotropin-releasing hormone (CRH)” refersto the polypeptide hormone and neurotransmitter (also known ascorticotropin-releasing factor (CRF) or corticoliberin). CRH is setforth e.g. in NP_000747.1.

The medical condition in which a low CRH level is therapeuticallybeneficial may comprise, for example, stress, depression, anxiety,stress, fatigue, impaired cognitive function, panic attack, compulsivebehavior, addiction, social phobia, sleep disorder, food relateddisorder, growth disorder, reproduction disorder and obesity.

As used herein the phrase “medical condition in which a low glutamatereceptor level is therapeutically beneficial” refers to a disease ordisorder in which decreasing the expression or activity of a glutamatereceptor can prevent an occurrence of a disease or medical symptomsassociated therewith or halt disease progression or medical symptomsassociated therewith (as further detailed hereinbelow).

As used herein, the term “glutamate receptor” refers to a synapticreceptor typically located on the membranes of neuronal cells (e.g.Grm1, Grik3, Grm5, Gria2, Grik2 and Grm7). Glutamate receptor is setforth e.g. in NP_000822.2 [glutamate receptor ionotropic kainate 3(Grik3)]; NP_000817.2, NP_001077088.1, NP_001077089.1 [glutamatereceptor ionotropic AMPA 2 (Gria2)]; NP_001159719.1, NP_068775.1,NP_786944.1 [glutamate receptor ionotropic kainate 2 (Grik2)];NP_000833.1, NP_001137303.1 [glutamate receptor metabotropic 5 (Grm5)];NP_000835.1, NP_870989.1 [glutamate receptor metabotropic 7 (Grm7)];NP_000829.2, NP_001107801.1 [glutamate receptor metabotropic 1 (Grm1)].

The medical condition in which a low glutamate receptor level istherapeutically beneficial may comprise, for example, seizures (e.g.epilepsy), Huntington's disease, Schizophrenia, Fragile X syndrome,generalized anxiety disorder and cancer (e.g. melanoma).

As used herein, the term “microRNA or a precursor thereof” refers to themicroRNA (miRNA) molecules acting as post-transcriptional regulators.MicroRNAs are typically processed from pre-miR (pre-microRNAprecursors). Pre-miRs are a set of precursor miRNA molecules transcribedby RNA polymerase III that are efficiently processed into functionalmiRNAs, e.g., upon transfection into cultured cells. A Pre-miR can beused to elicit specific miRNA activity in cell types that do notnormally express this miRNA, thus addressing the function of its targetby down regulating its expression in a “gain of (miRNA) function”experiment. Pre-miR designs exist to all of the known miRNAs listed inthe miRNA Registry and can be readily designed for any research. ThemicroRNAs may be administered to the cell per se or encoded from aprecursor molecule ligated into a nucleic acid construct, as furtherdescribed hereinbelow.

It will be appreciated that the microRNAs of the present teachings maybind, attach, regulate, process, interfere, augment, stabilize and/ordestabilize any microRNA target. Such a target can be any molecule,including, but not limited to, DNA molecules, RNA molecules andpolypeptides, such as but not limited to, serotonin related genes, suchas the serotonin transporter (i.e. SERT or Slc6a4), the serotonininhibitory receptor 1a (Htr1a), tryptophan hydroxylase 2 (Tph2) andmonoamine hydroxylase (MaoA); adrenaline or noradrenaline receptors(adrenergic receptors such as Add); Adenylate cyclase type 1 (ADCY1);CRH receptors such as Crh1R; or any other molecules e.g. FK506 bindingprotein 5 (FKBP5), canabinoid receptor 1 (CB1), Down Syndrome CellAdhesion Molecule (Dscam), Translin-associated protein X (Tsnax) andCell adhesion molecule L1 (L1cam), all described in further detailhereinbelow.

It will be appreciated that the microRNAs of the present invention canbe identified via various databases including for example the micro-RNAregistry(http://www(dot)sangerdotac(dot)uk/Software/Rfam/mirna/index(dot)shtml).

The methods of the present invention may be effected by administering toor expressing in a cell of the subject an exogenous polynucleotideencoding a microRNA.

The term “polynucleotide” refers to a single-stranded or double-strandedoligomer or polymer of ribonucleic acid (RNA), deoxyribonucleic acid(DNA) or mimetics thereof. This term includes polynucleotides and/oroligonucleotides derived from naturally occurring nucleic acidsmolecules (e.g., RNA or DNA), synthetic polynucleotide and/oroligonucleotide molecules composed of naturally occurring bases, sugars,and covalent internucleoside linkages (e.g., backbone), as well assynthetic polynucleotides and/or oligonucleotides having non-naturallyoccurring portions, which function similarly to respective naturallyoccurring portions.

The length of the polynucleotide of the present invention is optionallyof 100 nucleotides or less, optionally of 90 nucleotides or less,optionally 80 nucleotides or less, optionally 70 nucleotides or less,optionally 60 nucleotides or less, optionally 50 nucleotides or less,optionally 40 nucleotides or less, optionally 30 nucleotides or less,e.g., 29 nucleotides, 28 nucleotides, 27 nucleotides, 26 nucleotides, 25nucleotides, 24 nucleotides, 23 nucleotides, 22 nucleotides, 21nucleotides, 20 nucleotides, 19 nucleotides, 18 nucleotides, 17nucleotides, 16 nucleotides, 15 nucleotides, optionally between 12 and24 nucleotides, optionally between 5-15, optionally, between 5-25, mostpreferably, about 20-25 nucleotides.

The polynucleotides (including oligonucleotides) designed according tothe teachings of the present invention can be generated according to anyoligonucleotide synthesis method known in the art, including bothenzymatic syntheses or solid-phase syntheses. Equipment and reagents forexecuting solid-phase synthesis are commercially available from, forexample, Applied Biosystems. Any other means for such synthesis may alsobe employed; the actual synthesis of the oligonucleotides is well withinthe capabilities of one skilled in the art and can be accomplished viaestablished methodologies as detailed in, for example: Sambrook, J. andRussell, D. W. (2001), “Molecular Cloning: A Laboratory Manual”;Ausubel, R. M. et al., eds. (1994, 1989), “Current Protocols inMolecular Biology,” Volumes I-III, John Wiley & Sons, Baltimore, Md.;Perbal, B. (1988), “A Practical Guide to Molecular Cloning,” John Wiley& Sons, New York; and Gait, M. J., ed. (1984), “OligonucleotideSynthesis”; utilizing solid-phase chemistry, e.g. cyanoethylphosphoramidite followed by deprotection, desalting, and purificationby, for example, an automated trityl-on method or HPLC.

It will be appreciated that a polynucleotide comprising an RNA moleculecan be generated using an expression vector as is further describedhereinbelow.

Preferably, the polynucleotide of the present invention is a modifiedpolynucleotide. Polynucleotides can be modified using various methodsknown in the art.

For example, the oligonucleotides or polynucleotides of the presentinvention may comprise heterocylic nucleosides consisting of purines andthe pyrimidines bases, bonded in a 3′-to-5′ phosphodiester linkage.

Preferably used oligonucleotides or polynucleotides are those modifiedeither in backbone, internucleoside linkages, or bases, as is broadlydescribed hereinunder.

Specific examples of preferred oligonucleotides or polynucleotidesuseful according to this aspect of the present invention includeoligonucleotides or polynucleotides containing modified backbones ornon-natural internucleoside linkages. Oligonucleotides orpolynucleotides having modified backbones include those that retain aphosphorus atom in the backbone, as disclosed in U.S. Pat. Nos.4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

Preferred modified oligonucleotide or polynucleotide backbones include,for example: phosphorothioates; chiral phosphorothioates;phosphorodithioates; phosphotriesters; aminoalkyl phosphotriesters;methyl and other alkyl phosphonates, including 3′-alkylene phosphonatesand chiral phosphonates; phosphinates; phosphoramidates, including3′-amino phosphoramidate and aminoalkylphosphoramidates;thionophosphoramidates; thionoalkylphosphonates;thionoalkylphosphotriesters; and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogues of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts, and free acidforms of the above modifications can also be used.

Alternatively, modified oligonucleotide or polynucleotide backbones thatdo not include a phosphorus atom therein have backbones that are formedby short-chain alkyl or cycloalkyl internucleoside linkages, mixedheteroatom and alkyl or cycloalkyl internucleoside linkages, or one ormore short-chain heteroatomic or heterocyclic internucleoside linkages.These include those having morpholino linkages (formed in part from thesugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide,and sulfone backbones; formacetyl and thioformacetyl backbones;methylene formacetyl and thioformacetyl backbones; alkene-containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts, as disclosed inU.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141;5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677;5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240;5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070;5,663,312; 5,633,360; 5,677,437; and 5,677,439.

Other oligonucleotides or polynucleotides which may be used according tothe present invention are those modified in both sugar and theinternucleoside linkage, i.e., the backbone of the nucleotide units isreplaced with novel groups. The base units are maintained forcomplementation with the appropriate polynucleotide target. An exampleof such an oligonucleotide mimetic includes a peptide nucleic acid(PNA). A PNA oligonucleotide refers to an oligonucleotide where thesugar-backbone is replaced with an amide-containing backbone, inparticular an aminoethylglycine backbone. The bases are retained and arebound directly or indirectly to aza-nitrogen atoms of the amide portionof the backbone. United States patents that teach the preparation of PNAcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262; each of which is herein incorporated byreference. Other backbone modifications which may be used in the presentinvention are disclosed in U.S. Pat. No. 6,303,374.

Oligonucleotides or polynucleotides of the present invention may alsoinclude base modifications or substitutions. As used herein,“unmodified” or “natural” bases include the purine bases adenine (A) andguanine (G) and the pyrimidine bases thymine (T), cytosine (C), anduracil (U). “Modified” bases include but are not limited to othersynthetic and natural bases, such as: 5-methylcytosine (5-me-C);5-hydroxymethyl cytosine; xanthine; hypoxanthine; 2-aminoadenine;6-methyl and other alkyl derivatives of adenine and guanine; 2-propyland other alkyl derivatives of adenine and guanine; 2-thiouracil,2-thiothymine, and 2-thiocytosine; 5-halouracil and cytosine; 5-propynyluracil and cytosine; 6-azo uracil, cytosine, and thymine; 5-uracil(pseudouracil); 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl, and other 8-substituted adenines and guanines; 5-halo,particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracilsand cytosines; 7-methylguanine and 7-methyladenine; 8-azaguanine and8-azaadenine; 7-deazaguanine and 7-deazaadenine; and 3-deazaguanine and3-deazaadenine. Additional modified bases include those disclosed in:U.S. Pat. No. 3,687,808; Kroschwitz, J. I., ed. (1990), “The ConciseEncyclopedia Of Polymer Science And Engineering,” pages 858-859, JohnWiley & Sons; Englisch et al. (1991), “Angewandte Chemie,” InternationalEdition, 30, 613; and Sanghvi, Y. S., “Antisense Research andApplications,” Chapter 15, pages 289-302, S. T. Crooke and B. Lebleu,eds., CRC Press, 1993. Such modified bases are particularly useful forincreasing the binding affinity of the oligomeric compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines,and N-2, N-6, and O-6-substituted purines, including2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S. et al. (1993),“Antisense Research and Applications,” pages 276-278, CRC Press, BocaRaton), and are presently preferred base substitutions, even moreparticularly when combined with 2′-O-methoxyethyl sugar modifications.

According to a specific embodiment, the miRNA polynucleotide of thepresent invention has a nucleic acid sequence as set forth in SEQ IDNOs: 58-94 (see Table 1A).

TABLE 1A miRNA polynucleotide sequences Sequence miRNA SEQ ID NOs: 77-80miR-15 SEQ ID NOs: 72-76 miR-19 SEQ ID NOs: 65-69 miR-26 SEQ ID NOs:81-84 miR-27 SEQ ID NOs: 58-62 miR-135 SEQ ID NOs: 85-94 miR-181 SEQ IDNOs: 70-71 miR-182 SEQ ID NOs: 63-64 miR-335

As is mentioned hereinabove and is shown in the Examples section whichfollows, micro-RNAs are processed molecules derived from specificprecursors (i.e., pre-miRNA), upregulation of a specific miRNA functioncan be effected using a specific miRNA precursor molecule.

Also contemplated are sequences homologous to the miRNAs and precursorsthereof. The level of homology should be relatively high for the maturemiRNA but more orders of freedom are allowed at the precursor level(e.g., at least 60%, 70%, 80%, 85%, 90%, 95% or more) as long as thesequence alterations are in the hair pin sequence and not in the nucleicacid segment corresponding to the mature miR.

Such precursor polynucleotide agents are typically administered to thetarget cells (e.g. neuroglia cells or cardiac cells) as part of anexpression construct. In this case, the polynucleotide agent is ligatedin a nucleic acid construct under the control of a cis-acting regulatoryelement (e.g. promoter) capable of directing an expression of themicroRNA in the target cells (e.g. neuroglia cells or cardiac cells) ina constitutive or inducible manner.

Examples of microRNA polynucleotide agents of the present inventioninclude, but are not limited to, miR-15 (e.g. GenBank accession no.NR_029485 RNA), miR-19 (e.g. GenBank accession no. NR_029489.1), miR-26(e.g. GenBank accession nos. NR_029500 and NR_029499), miR-27 (e.g.GenBank accession no. NR_029501 RNA), miR-135 (e.g. GenBank accessionno. NR_029677.1), miR-335 (e.g. GenBank accession no. NR_029899.1),miR-181 (e.g. GenBank accession no. NR_029611.1) and miR-182 (e.g.GenBank accession no. NR_029614).

Examples of neuron cell specific promoters include, but are not limitedto, neuron-specific enolase gene promoter, synapsin promoter, enhancedsynapsin promoter, calcium calmodulin promoter and Thy1 promoter.

Examples of cardiac cell specific promoters include, but are not limitedto, cardiac NCX1 promoter and α-myosin heavy chain (αMHC) promoter.

The expression constructs of the present invention may also includeadditional sequences which render it suitable for replication andintegration in eukaryotes (e.g., shuttle vectors). Typical cloningvectors contain transcription and translation initiation sequences(e.g., promoters, enhances) and transcription and translationterminators (e.g., polyadenylation signals). The expression constructsof the present invention can further include an enhancer, which can beadjacent or distant to the promoter sequence and can function in upregulating the transcription therefrom.

Enhancer elements can stimulate transcription up to 1,000-fold fromlinked homologous or heterologous promoters. Enhancers are active whenplaced downstream or upstream from the transcription initiation site.Many enhancer elements derived from viruses have a broad host range andare active in a variety of tissues. For example, the SV40 early geneenhancer is suitable for many cell types. Other enhancer/promotercombinations that are suitable for the present invention include thosederived from polyoma virus or human or murine cytomegalovirus (CMV) andthe long tandem repeats (LTRs) from various retroviruses, such as murineleukemia virus, murine or Rous sarcoma virus, and HIV. See Gluzman, Y.and Shenk, T., eds. (1983). Enhancers and Eukaryotic Gene Expression,Cold Spring Harbor Press, Cold Spring Harbor, N.Y., which isincorporated herein by reference.

Polyadenylation sequences can also be added to the expression constructsof the present invention in order to increase the efficiency ofexpression of the detectable moeity. Two distinct sequence elements arerequired for accurate and efficient polyadenylation: GU- or U-richsequences located downstream from the polyadenylation site and a highlyconserved sequence of six nucleotides, namely AAUAAA, located 11-30nucleotides upstream of the site. Termination and polyadenylationsignals suitable for the present invention include those derived fromSV40.

In addition to the embodiments already described, the expressionconstructs of the present invention may typically contain otherspecialized elements intended to increase the level of expression ofcloned nucleic acids or to facilitate the identification of cells thatcarry the recombinant DNA. For example, a number of animal virusescontain DNA sequences that promote extra-chromosomal replication of theviral genome in permissive cell types. Plasmids bearing these viralreplicons are replicated episomally as long as the appropriate factorsare provided by genes either carried on the plasmid or with the genomeof the host cell.

The expression constructs of the present invention may or may notinclude a eukaryotic replicon. If a eukaryotic replicon is present, thevector is capable of amplification in eukaryotic cells using theappropriate selectable marker. If the construct does not comprise aeukaryotic replicon, no episomal amplification is possible. Instead, therecombinant DNA integrates into the genome of the engineered cell, wherethe promoter directs expression of the desired nucleic acid.

The nucleic acid construct may be introduced into the target cells (e.g.neuroglia cells or cardiac cells) of the present invention using anappropriate gene delivery vehicle/method (transfection, transduction,etc.) and an appropriate expression system. Examples of mammalianexpression vectors include, but are not limited to, pcDNA3,pcDNA3.1(+/−), pGL3, pZeoSV2(+/−), pSecTag2, pDisplay, pEF/myc/cyto,pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, and pNMT81,which are available from Invitrogen, pCI which is available fromPromega, pMbac, pPbac, pBK-RSV and pBK-CMV, which are available fromStrategene, pTRES which is available from Clontech, and theirderivatives.

Expression vectors containing regulatory elements from eukaryoticviruses such as retroviruses can be also used. SV40 vectors includepSVT7 and pMT2, for instance. Vectors derived from bovine papillomavirus include pBV-1MTHA, and vectors derived from Epstein-Barr virusinclude pHEBO and p205. Other exemplary vectors include pMSG, pAV009/A⁺,pMT010/A⁺, pMAMneo-5 and baculovirus pDSVE.

Lipid-based systems may be used for the delivery of these constructsinto the target cells (e.g. neuroglia cells or cardiac cells) of thepresent invention.

Liposomes include any synthetic (i.e., not naturally occurring)structure composed of lipid bilayers, which enclose a volume. Liposomesinclude emulsions, foams, micelles, insoluble monolayers, liquidcrystals, phospholipid dispersions, lamellar layers and the like. Theliposomes may be prepared by any of the known methods in the art[Monkkonen, J. et al., 1994, J. Drug Target, 2:299-308; Monkkonen, J. etal., 1993, Calcif. Tissue Int., 53:139-145; Lasic D D., LiposomesTechnology Inc., Elsevier, 1993, 63-105. (chapter 3); Winterhalter M,Lasic D D, Chem Phys Lipids, 1993 September; 64(1-3):35-43]. Theliposomes may be positively charged, neutral or negatively charged. ForMononuclear Phagocyte System (MPS) uptake, the liposomes can behydrophobic since hydrophilic masking of the liposome membrane (e.g., byuse of polyetheleneglycol-linked lipids and hydrophilic particles) maybe less prone to MPS uptake. It is also preferable that the liposomes donot comprise sterically shielded lipids such as ganglioside-GM1 andphosphatidylinositol since these lipids prevent MPS uptake.

The liposomes may be a single lipid layer or may be multilamellar. Ifthe therapeutic agent is hydrophilic, its delivery may be furtherimproved using large unilamellar vesicles because of their greaterinternal volume. Conversely, if the therapeutic agent is hydrophobic,its delivery may be further improved using multilamellar vesicles.Alternatively, the therapeutic agent (e.g. oligonucleotide) may not beable to penetrate the lipid bilayer and consequently would remainadsorbed to the liposome surface. In this case, increasing the surfacearea of the liposome may further improve delivery of the therapeuticagent. Suitable liposomes in accordance with the invention are non-toxicliposomes such as, for example, those prepared from phosphatidyl-cholinephosphoglycerol, and cholesterol. The diameter of the liposomes used canrange from 0.1-1.0 microns. However, other size ranges suitable forphagocytosis by phagocytic cells may also be used. For sizing liposomes,homogenization may be used, which relies on shearing energy to fragmentlarge liposomes into smaller ones. Homogenizers which may beconveniently used include microfluidizers produced by Microfluidics ofBoston, Mass. In a typical homogenization procedure, liposomes arerecirculated through a standard emulsion homogenizer until selectedliposomes sizes are observed. The particle size distribution can bemonitored by conventional laser beam particle size discrimination.Extrusion of liposomes through a small-pore polycarbonate membrane or anasymmetric ceramic membrane is an effective method for reducing liposomesizes to a relatively well defined size distribution. Typically, thesuspension is cycled through the membrane one or more times until thedesired liposome size distribution is achieved. The liposomes may beextruded through successively smaller pore membranes to achieve agradual reduction in liposome size.

Any method known in the art can be used to incorporate micro-RNApolynucleotide agent into a liposome. For example, the micro-RNApolynucleotide agent may be encapsulated within the liposome.Alternatively, it may be adsorbed on the liposome's surface. Othermethods that may be used to incorporate a pharmaceutical agent into aliposome of the present invention are those described by Alfonso et al.,[The science and practice of pharmacy, Mack Publishing, Easton Pa.19^(th) ed., (1995)] and those described by Kulkarni et al., [J.Microencapsul. 1995, 12 (3) 229-46].

The liposomes used in the methods of the present invention preferablycross the blood barriers. Thus, the liposomes of the present inventionpreferably do not comprise a blood barrier targeting polysaccharide(e.g. mannose) in their membrane portion. Preferably, the liposomes ofthe present invention do not comprise peptides in their membrane portionthat target the liposomes to a receptor on a blood barrier. Examples ofsuch peptides include but are not limited to transferrin, insulin,IGF-1, IGF-2 anti-transferrin receptor antibody, anti-insulin receptorantibody, anti-IGF-1 receptor antibody and anti-IGF-2 receptor antibody.

In order to determine liposomes that are especially suitable inaccordance with the present invention a screening assay can be performedsuch as the assays described in U.S. Pat. Appl. No. 20040266734 and U.S.Pat. Appl. No. 20040266734; and in Danenberg et al., Journal ofcardiovascular pharmacology 2003, 42:671-9; Circulation 2002,106:599-605; Circulation 2003, 108:2798-804.

Other non-lipid based vectors that can be used according to this aspectof the present invention include but are not limited to polylysine anddendrimers.

The expression construct may also be a virus. Examples of viralconstructs include but are not limited to adenoviral vectors, retroviralvectors, vaccinia viral vectors, adeno-associated viral vectors, polyomaviral vectors, alphaviral vectors, rhabdoviral vectors, lenti viralvectors and herpesviral vectors.

Retroviral vectors represent a class of vectors particularly suitablefor use with the present invention. Defective retroviruses are routinelyused in transfer of genes into mammalian cells (for a review, seeMiller, A. D. (1990). Blood 76, 271). A recombinant retroviruscomprising the polynucleotides of the present invention can beconstructed using well-known molecular techniques. Portions of theretroviral genome can be removed to render the retrovirus replicationmachinery defective, and the replication-deficient retrovirus can thenpackaged into virions, which can be used to infect target cells throughthe use of a helper virus while employing standard techniques. Protocolsfor producing recombinant retroviruses and for infecting cells withviruses in vitro or in vivo can be found in, for example, Ausubel et al.(1994) Current Protocols in Molecular Biology (Greene PublishingAssociates, Inc. & John Wiley & Sons, Inc.). Retroviruses have been usedto introduce a variety of genes into many different cell types,including neuronal cells, epithelial cells, endothelial cells,lymphocytes, myoblasts, hepatocytes, and bone marrow cells.

According to one embodiment, a lentiviral vector, a type of retroviralvector, is used according to the present teachings. Lentiviral vectorsare widely used as vectors due to their ability to integrate into thegenome of non-dividing as well as dividing cells. The viral genome, inthe form of RNA, is reverse-transcribed when the virus enters the cellto produce DNA, which is then inserted into the genome at a randomposition by the viral integrase enzyme. The vector (a provirus) remainsin the genome and is passed on to the progeny of the cell when itdivides. For safety reasons, lentiviral vectors never carry the genesrequired for their replication. To produce a lentivirus, severalplasmids are transfected into a so-called packaging cell line, commonlyHEK 293. One or more plasmids, generally referred to as packagingplasmids, encode the virion proteins, such as the capsid and the reversetranscriptase. Another plasmid contains the genetic material to bedelivered by the vector. It is transcribed to produce thesingle-stranded RNA viral genome and is marked by the presence of the w(psi) sequence. This sequence is used to package the genome into thevirion.

A specific example of a suitable lentiviral vector for introducing andexpressing the polynucleotide sequences of the present invention inneuroglia cells or cardiac cells is the lentivirus pLKO.1 vector.

Another suitable expression vector that may be used according to thisaspect of the present invention is the adenovirus vector. The adenovirusis an extensively studied and routinely used gene transfer vector. Keyadvantages of an adenovirus vector include relatively high transductionefficiency of dividing and quiescent cells, natural tropism to a widerange of epithelial tissues, and easy production of high titers (Russel,W. C. (2000) J Gen Virol 81, 57-63). The adenovirus DNA is transportedto the nucleus, but does not integrate thereinto. Thus the risk ofmutagenesis with adenoviral vectors is minimized, while short-termexpression is particularly suitable for treating cancer cells.Adenoviral vectors used in experimental cancer treatments are describedby Seth et al. (1999). “Adenoviral vectors for cancer gene therapy,” pp.103-120, P. Seth, ed., Adenoviruses: Basic Biology to Gene Therapy,Landes, Austin, Tex.).

A suitable viral expression vector may also be a chimericadenovirus/retrovirus vector combining retroviral and adenoviralcomponents. Such vectors may be more efficient than traditionalexpression vectors for transducing tumor cells (Pan et al. (2002).Cancer Letts 184, 179-188).

When introducing the expression constructs of the present invention intotarget cells (e.g. neuroglia cells or cardiac cells) by viral infectionthe viral dose for infection is at least 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸,10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵ or higher pfu or viralparticles.

Regardless of the method or construct employed, there is provided anisolated cell comprising the nucleic acid construct encoding a microRNA,as detailed above.

As used herein the term “isolated” refers to at least partiallyseparated from the natural environment e.g., the human body.

According to one embodiment, there is provided an isolated cellcomprising a nucleic acid construct expressing at least one microRNA ora precursor thereof, wherein the microRNA is selected from the groupconsisting of miR-135, miR-335, miR-15, miR-19, miR-26, miR-27, miR-181and miR-182 under a transcriptional control of a cis acting regulatoryelement.

According to a specific embodiment, there is provided an isolatedneuroglia cell comprising a nucleic acid construct expressing at leastone microRNA or a precursor thereof, wherein the microRNA is selectedfrom the group consisting of miR-135, miR-335, miR-26 and miR-182 undera transcriptional control of a cis acting regulatory element.

According to a specific embodiment, there is provided an isolated cellcomprising a nucleic acid construct expressing a miR-19 or a precursorthereof under a transcriptional control of a cis acting regulatoryelement.

According to a specific embodiment, there is provided an isolated cellcomprising a nucleic acid construct expressing a miR-15 or a precursorthereof under a transcriptional control of a cis acting regulatoryelement.

According to a specific embodiment, the cell is a neuroglia cell or acardiac cell. According to a specific embodiment, the neuroglia cell isa neuron such as a serotonergic neuron.

The microRNAs or precursors thereof are to be provided to the cellsi.e., target cells (e.g. neuroglia cells or cardiac cells) of thepresent invention in vivo (i.e., inside the organism or the subject) orex vivo (e.g., in a tissue culture). In case the cells are treated exvivo, the method preferably includes a step of administering such cellsback to the individual (ex vivo cell therapy).

For ex vivo therapy, cells are preferably treated with the agent of thepresent invention (e.g., a polynucleotide encoding a microRNA),following which they are administered to the subject in need thereof.

Administration of the ex vivo treated cells of the present invention canbe effected using any suitable route of introduction, such asintravenous, intraperitoneal, intra-kidney, intra-gastrointestinaltrack, subcutaneous, transcutaneous, intramuscular, intracutaneous,intrathecal, epidural, and rectal. According to presently preferredembodiments, the ex vivo treated cells of the present invention may beintroduced to the individual using intravenous, intra-kidney,intra-gastrointestinal track, and/or intraperitoneal administration.

The cells of the present invention (e.g. neuroglia cells or cardiaccells) can be derived from either autologous sources or from allogeneicsources such as human cadavers or donors. Since non-autologous cells arelikely to induce an immune reaction when administered to the bodyseveral approaches have been developed to reduce the likelihood ofrejection of non-autologous cells. These include either suppressing therecipient immune system or encapsulating the non-autologous cells inimmunoisolating, semipermeable membranes before transplantation.

Encapsulation techniques are generally classified as microencapsulation,involving small spherical vehicles, and macroencapsulation, involvinglarger flat-sheet and hollow-fiber membranes (Uludag, H. et al. (2000).Technology of mammalian cell encapsulation. Adv Drug Deliv Rev 42,29-64).

Methods of preparing microcapsules are known in the art and include forexample those disclosed in: Lu, M. Z. et al. (2000). Cell encapsulationwith alginate and alpha-phenoxycinnamylidene-acetylatedpoly(allylamine). Biotechnol Bioeng 70, 479-483; Chang, T. M. andPrakash, S. (2001) Procedures for microencapsulation of enzymes, cellsand genetically engineered microorganisms. Mol Biotechnol 17, 249-260;and Lu, M. Z., et al. (2000). A novel cell encapsulation method usingphotosensitive poly(allylamine alpha-cyanocinnamylideneacetate). JMicroencapsul 17, 245-521.

For example, microcapsules are prepared using modified collagen in acomplex with a ter-polymer shell of 2-hydroxyethyl methylacrylate(HEMA), methacrylic acid (MAA), and methyl methacrylate (MMA), resultingin a capsule thickness of 2-5 μm. Such microcapsules can be furtherencapsulated with an additional 2-5 μm of ter-polymer shells in order toimpart a negatively charged smooth surface and to minimize plasmaprotein absorption (Chia, S. M. et al. (2002). Multi-layeredmicrocapsules for cell encapsulation. Biomaterials 23, 849-856).

Other microcapsules are based on alginate, a marine polysaccharide(Sambanis, A. (2003). Encapsulated islets in diabetes treatment.Diabetes Thechnol Ther 5, 665-668), or its derivatives. For example,microcapsules can be prepared by the polyelectrolyte complexationbetween the polyanions sodium alginate and sodium cellulose sulphate andthe polycation poly(methylene-co-guanidine) hydrochloride in thepresence of calcium chloride.

It will be appreciated that cell encapsulation is improved when smallercapsules are used. Thus, for instance, the quality control, mechanicalstability, diffusion properties, and in vitro activities of encapsulatedcells improved when the capsule size was reduced from 1 mm to 400 μm(Canaple, L. et al. (2002). Improving cell encapsulation through sizecontrol. J Biomater Sci Polym Ed 13, 783-96). Moreover, nanoporousbiocapsules with well-controlled pore size as small as 7 nm, tailoredsurface chemistries, and precise microarchitectures were found tosuccessfully immunoisolate microenvironments for cells (See: Williams,D. (1999). Small is beautiful: microparticle and nanoparticle technologyin medical devices. Med Device Technol 10, 6-9; and Desai, T. A. (2002).Microfabrication technology for pancreatic cell encapsulation. ExpertOpin Biol Ther 2, 633-646).

Examples of immunosuppressive agents which may be used in conjunctionwith the ex vivo treatment include, but are not limited to,methotrexate, cyclophosphamide, cyclosporine, cyclosporin A,chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine),gold salts, D-penicillamine, leflunomide, azathioprine, anakinra,infliximab (REMICADE.sup.R), etanercept, TNF.alpha. blockers, abiological agent that targets an inflammatory cytokine, andNon-Steroidal Anti-Inflammatory Drug (NSAIDs). Examples of NSAIDsinclude, but are not limited to acetyl salicylic acid, choline magnesiumsalicylate, diflunisal, magnesium salicylate, salsalate, sodiumsalicylate, diclofenac, etodolac, fenoprofen, flurbiprofen,indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen,nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin,acetaminophen, ibuprofen, Cox-2 inhibitors and tramadol.

For in vivo therapy, the agent (e.g., a polynucleotide encoding amicroRNA) is administered to the subject per se or as part of apharmaceutical composition. Preferably such compositions are formulatedto allow passage through the blood brain barrier (BBB).

Conventional approaches for drug delivery to the central nervous system(CNS) include: neurosurgical strategies (e.g., intracerebral injectionor intracerebroventricular infusion); molecular manipulation of theagent (e.g., production of a chimeric fusion protein that comprises atransport peptide that has an affinity for an endothelial cell surfacemolecule in combination with an agent that is itself incapable ofcrossing the BBB) in an attempt to exploit one of the endogenoustransport pathways of the BBB; pharmacological strategies designed toincrease the lipid solubility of an agent (e.g., conjugation ofwater-soluble agents to lipid or cholesterol carriers); and thetransitory disruption of the integrity of the BBB by hyperosmoticdisruption (resulting from the infusion of a mannitol solution into thecarotid artery or the use of a biologically active agent such as anangiotensin peptide).

Methods for drug delivery behind the BBB include intracerebralimplantation (such as with needles) and convection-enhanceddistribution. Mannitol can be used in bypassing the BBB. Likewise,mucosal (e.g., nasal) administration can be used to bypass the BBB.

The micro-RNA polynucleotide agents of the present invention can also beadministered to an organism in a pharmaceutical composition where it ismixed with suitable carriers or excipients.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the peptide accountablefor the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington' s Pharmaceutical Sciences,” Mack Publishing Co., Easton,Pa., latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients (peptide) effective to prevent, alleviate orameliorate symptoms of a disorder (e.g., diabetes) or prolong thesurvival of the subject being treated.

According to an embodiment of the present invention, overexpression ofmiR-135 has an anti-depressant effect.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein. For any preparation used in themethods of the invention, the therapeutically effective amount or dosecan be estimated initially from in vitro and cell culture assays. Forexample, a dose can be formulated in animal models to achieve a desiredconcentration or titer. Such information can be used to more accuratelydetermine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p.1).

Dosage amount and interval may be adjusted individually to providesufficient plasma levels of the active ingredient to induce or suppressthe biological effect (minimal effective concentration, MEC). The MECwill vary for each preparation, but can be estimated from in vitro data.Dosages necessary to achieve the MEC will depend on individualcharacteristics and route of administration. Detection assays can beused to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc. The dosage and timing of administration will be responsive to acareful and continuous monitoring of the individual changing condition.

It will be appreciated that animal models exist by which the agents ofthe present invention may be tested prior to human treatment. Forexample, animal models of depression, stress, anxiety such as learnedhelplessness model (LH), chronic mild stress (CMS) model, social defeatstress (SDS) model and maternal deprivation model may be used.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as is further detailed above.

It will be appreciated that the therapeutic compositions of theinvention may comprise, in addition to the micro-RNA polynucleotideagents, other known medications for the treatment of depression, stress,anxiety, sleep deprivation, etc. such as, but not limited to, selectiveserotonin reuptake inhibitors (SSRIs), serotonin-norepinephrine reuptakeinhibitors (SNRIs), noradrenergic and specific serotonergicantidepressants (NaSSAs), norepinephrine (noradrenaline) reuptakeinhibitors (NRIs), norepinephrine-dopamine reuptake inhibitors,selective serotonin reuptake enhancers, norepinephrine-dopaminedisinhibitors, tricyclic antidepressants (e.g. Imipramine), monoamineoxidase inhibitors (MAOIs). These medications may be included in thearticle of manufacture in a single or in separate packagings.

The present inventors have shown that overexpression of miR-27 resultsin suppression of MaoA (see Example 1, hereinbelow), overexpression ofmiR-135 results in suppression of Slc6a4 (see Example 1, hereinbelow),overexpression of miR-135, miR-335, miR-26, miR-181 or miR-182 resultsin suppression of Htr1a (see Example 1, hereinbelow), overexpression ofmiR-19 results in suppression of Add (see Example 2, hereinbelow) and insuppression of CB1 (see Example 3B, hereinbelow), and thatoverexpression of miR-15 results in suppression of Crh1R (see Example 4,hereinbelow) and in suppression of FKBP5 (see Example 4B, hereinbelow).

Thus, according to one embodiment of the present invention, there isprovided a method of regulating an expression of a serotonin transporter(Slc6a4) gene in a neuroglia cell, the method comprising modulating anactivity or expression of a microRNA or a precursor thereof, wherein themicroRNA is selected from the group consisting of miR-135 and miR-335.

As used herein, the term “serotonin transporter (Slc6a4)” refers to themonoamine transporter protein (also named SERT) involved in reuptake ofserotonin from the synaptic cleft. An exemplary Slc6a4 is set forth inNP_001036.1.

According to another embodiment, there is provided a method ofregulating an expression of a serotonin inhibitory receptor 1a (Htr1a)gene in a neuroglia cell, the method comprising modulating an activityor expression of a microRNA or a precursor thereof in the neurogliacell, wherein the microRNA is selected from the group consisting ofmiR-135, miR-335, miR-181, miR-182 and miR-26.

As used herein, the term “serotonin inhibitory receptor 1a (Htr1a)”refers to the G protein-coupled receptor that functions as anautoreceptor in the presynaptic neuron and mediated inhibition ofserotonin release. An exemplary Htr1a is set forth in NP_000515.2.

According to another embodiment, there is provided a method ofregulating an expression of a monoamine hydroxylase (MaoA) gene in aneuroglia cell, the method comprising modulating an activity orexpression of a miR-27 or a precursor thereof.

As used herein, the term “monoamine hydroxylase (MaoA)” refers to theenzyme that degrades amine neurotransmitters, such as dopamine,norepinephrine, and serotonin. An exemplary MaoA is set forth inNP_000231.1.

According to one embodiment of the present invention, there is provideda method of regulating an expression of a tryptophan hydroxylase 2(Tph2) gene in a neuroglia cell, the method comprising modulating anactivity or expression of a microRNA or a precursor thereof in theneuroglia cell, wherein the microRNA is selected from the groupconsisting of miR-181 and miR27.

As used herein, the term “tryptophan hydroxylase 2 (Tph2)” refers to theenzyme which catalyzes the first and rate limiting step in thebiosynthesis of serotonin. In exemplary Tph2 is set forth inNP_NP_775489.2.

According to another embodiment, there is provided a method ofregulating an expression of a beta adrenergic receptor 1 (Adrb1) gene ina neuroglia cell or cardiac cell, the method comprising modulating anactivity or expression of a miR-19 or a precursor thereof.

As used herein, the term “beta adrenergic receptor 1 (Adrb1)” refers tothe receptor that mediates the physiological effects of adrenaline andnoradrenaline. An exemplary Adrb1 is set forth in NP_000675.1.

According to another embodiment, there is provided a method ofregulating an expression of a beta 2 adrenergic receptor (Adrb2) gene ina neuroglia cell, the method comprising modulating an activity orexpression of a miR-15 or a precursor thereof.

As used herein, the term “beta 2 adrenergic receptor (Adrb2)” refers tothe receptor that is directly associated with the class C L-type calciumchannel Ca(V)1.2. Adrb2 is set forth e.g. in NP_000015.1.

According to another embodiment, there is provided a method ofregulating an expression of a CRH type 1 receptor gene in a neurogliacell, the method comprising modulating an activity or expression of amiR-15 or a precursor thereof.

As used herein, the term “CRH type 1” refers to the receptor which bindscorticotropin-releasing hormone (CRH). CRH type 1 is set forth e.g. inNP_001138618.1, NP_001138619.1, NP_001138620.1 and NP_004373.2.

According to another embodiment, there is provided a method ofregulating an expression of a glutamate receptor gene in a neurogliacell, the method comprising modulating an activity or expression ofmiR-181 or a precursor thereof.

According to another embodiment, the glutamate receptor gene comprisesglutamate receptor metabotropic 1 (Grm1), glutamate receptor ionotropickainate 3 (Grik3), glutamate receptor metabotropic 5 (Grm5), glutamatereceptor ionotropic kainate 2 (Grik2) and glutamate receptormetabotropic 7 (Grm7), as described in further detail above.

According to another embodiment, there is provided a method ofregulating an expression of a Down Syndrome Cell Adhesion Molecule(Dscam) gene in a neuroglia cell, the method comprising modulating anactivity or expression of a miR-182 or a precursor thereof.

As used herein, the term “Down Syndrome Cell Adhesion Molecule (Dscam)”refers to the cell adhesion molecule that plays a role in neuronalself-avoidance. Dscam is set forth e.g. in NP_001380.2.

According to another embodiment, there is provided a method ofregulating an expression of a Cell adhesion molecule L1 (L1cam) gene ina neuroglia cell, the method comprising modulating an activity orexpression of a miR-182 or a precursor thereof.

As used herein, the term “Cell adhesion molecule L1 (L1cam)” refers tothe neuronal cell adhesion molecule. L1cam is set forth e.g. inNP_000416.1, NP_001137435.1, NP_076493.1.

According to another embodiment, there is provided a method ofregulating an expression of a Translin-associated protein X (Tsnax) genein a neuroglia cell, the method comprising modulating an activity orexpression of a miR-182 or a precursor thereof.

As used herein, the term “Translin-associated protein X (Tsnax)” refersto the protein which specifically interacts with translin. Tsnax is setforth e.g. in NP_005990.1.

According to another embodiment, there is provided a method ofregulating an expression of a canabinoid receptor 1 (CB1) gene in aneuroglia cell, the method comprising modulating an activity orexpression of a miR-19 or a precursor thereof.

As used herein, the term “canabinoid receptor 1 (CB1)” refers to the ofcell membrane receptor (also known as CNR1). CB1 is set forth e.g. inNP_001153698.1, NP_001153730.1, NP_001153731.1, NP_057167.2,NP_149421.2.

According to another embodiment, there is provided a method ofregulating an expression of a FK506 binding protein 5 (FKBP5) gene in aneuroglia cell, the method comprising modulating an activity orexpression of a miR-15 or a precursor thereof.

As used herein, the term “FK506 binding protein 5 (FKBP5)” refers to theprotein which specifically binds to the immunosuppressants FK506 andrapamycin. FKBP5 is set forth e.g. in NP_001139247.1, NP_001139248.1,NP_001139249.1, NP_004108.1.

According to another embodiment, there is provided a method ofregulating an expression of a syntaxin 1a (Stx1a) gene in a neurogliacell, the method comprising modulating an activity or expression of amiR-15 or a precursor thereof.

As used herein, the term “syntaxin 1a (Stx1a)” refers to the nervoussystem-specific protein. Stx1a is set forth e.g. in NP_001159375.1,NP_004594.1.

According to another embodiment, there is provided a method ofregulating an expression of a serum/glucocorticoid regulated kinase(Sgk1) gene in a neuroglia cell, the method comprising modulating anactivity or expression of a miR-15 or a precursor thereof.

As used herein, the term “serum/glucocorticoid regulated kinase (Sgk1)”refers to serine/threonine protein kinase. Sgk1 is set forth e.g. inNP_001137148.1, NP_001137149.1, NP_001137150.1, NP_005618.2.

The present teachings contemplate upregulating (i.e. increasing) ordownregulating (i.e. decreasing) the expression levels of theaforementioned genes.

Downregulation of gene expression according to the present teachings istypically carried out by administering to or expressing in the targetcells (e.g. neuroglia cell or cardiac cell) a microRNA polynucleotide(as depicted in further detail hereinabove).

According to a specific embodiment, when the regulating comprisesdownregulating the expression of the Slc6a4 gene, the modulatingcomprises upregulating the miR-135 and/or miR-335.

According to a specific embodiment, when the regulating comprisesdownregulating the expression of the Htr1a gene, the modulatingcomprises upregulating the miR-135, miR-335, miR-181, miR-182 and/ormiR-26.

According to a specific embodiment, when the regulating comprisesdownregulating the expression of the MaoA gene, the modulating comprisesupregulating the miR-27.

According to a specific embodiment, when the regulating comprisesdownregulating the expression of the Adrb1 gene, the modulatingcomprises upregulating the miR-19.

According to a specific embodiment, when the regulating comprisesdownregulating the expression of the CRH type 1 receptor gene, themodulating comprises upregulating the miR-15.

According to a specific embodiment, when the regulating comprisesdownregulating the expression of the CB1 gene, the modulating comprisesupregulating the miR-19.

According to a specific embodiment, when the regulating comprisesdownregulating the expression of the FKBP5 gene, the modulatingcomprises upregulating the miR-15.

Alternatively, according to another embodiment of the present invention,upregulating gene expression is affected by administering to orexpressing in the target cells (e.g. neuroglia cell or cardiac cell) anagent capable of downregulating an expression of a microRNA.

Downregulation of microRNAs can be effected on the genomic and/or thetranscript level using a variety of molecules which interfere withtranscription and/or translation (e.g., RNA silencing agents, Ribozyme,DNAzyme and antisense).

Methods of downregulating microRNA expression are known in the art.

Nucleic acid agents that down-regulate miR activity include, but are notlimited to, a target mimic, a micro-RNA resistant gene and a miRNAinhibitor.

The target mimic or micro-RNA resistant target is essentiallycomplementary to the microRNA provided that one or more of followingmismatches are allowed:

(a) a mismatch between the nucleotide at the 5′ end of the microRNA andthe corresponding nucleotide sequence in the target mimic or micro-RNAresistant target;

(b) a mismatch between any one of the nucleotides in position 1 toposition 9 of the microRNA and the corresponding nucleotide sequence inthe target mimic or micro-RNA resistant target; or

(c) three mismatches between any one of the nucleotides in position 12to position 21 of the microRNA and the corresponding nucleotide sequencein the target mimic or micro-RNA resistant target provided that thereare no more than two consecutive mismatches.

The target mimic RNA is essentially similar to the target RNA modifiedto render it resistant to miRNA induced cleavage, e.g. by modifying thesequence thereof such that a variation is introduced in the nucleotideof the target sequence complementary to the nucleotides 10 or 11 of themiRNA resulting in a mismatch.

Alternatively, a microRNA-resistant target may be implemented. Thus, asilent mutation may be introduced in the microRNA binding site of thetarget gene so that the DNA and resulting RNA sequences are changed in away that prevents microRNA binding, but the amino acid sequence of theprotein is unchanged. Thus, a new sequence can be synthesized instead ofthe existing binding site, in which the DNA sequence is changed,resulting in lack of miRNA binding to its target.

According to a specific embodiment, the target mimic or micro-RNAresistant target is linked to the promoter naturally associated with thepre-miRNA recognizing the target gene and introduced into the plantcell. In this way, the miRNA target mimic or micro-RNA resistant targetRNA will be expressed under the same circumstances as the miRNA and thetarget mimic or micro-RNA resistant target RNA will substitute for thenon-target mimic/micro-RNA resistant target RNA degraded by the miRNAinduced cleavage.

Non-functional miRNA alleles or miRNA resistant target genes may also beintroduced by homologous recombination to substitute the miRNA encodingalleles or miRNA sensitive target genes.

Recombinant expression is effected by cloning the nucleic acid ofinterest (e.g., miRNA, target gene, silencing agent etc) into a nucleicacid expression construct under the expression of a plant promoter.

In other embodiments of the invention, synthetic single stranded nucleicacids are used as miRNA inhibitors. A miRNA inhibitor is typicallybetween about 17 to 25 nucleotides in length and comprises a 5′ to 3′sequence that is at least 90% complementary to the 5′ to 3′ sequence ofa mature miRNA. In certain embodiments, a miRNA inhibitor molecule is17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, or anyrange derivable therein. Moreover, a miRNA inhibitor has a sequence(from 5′ to 3′) that is or is at least 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100%complementary, or any range derivable therein, to the 5′ to 3′ sequenceof a mature miRNA, particularly a mature, naturally occurring miRNA.

The miRNA inhibitors may be contacted with the cells using transienttransfection techniques. miRNA inhibitors are commercially availablefrom Companies such as Applied Biosystems.

Alternatively, the miRNA inhibitors may be part of an expression vector,as described herein above. In this case, cells may be transiently orstably transfected with the vector.

According to a specific embodiment, when the regulating comprisesupregulating the expression of the Tph2 gene, the modulating comprisesdownregulating the miR-181 and/or miR-27.

According to one embodiment, downregulating the expression of a microRNAis effected by the use of a nucleic acid sequence which specificallybinds and downregulates the expression of the microRNA. An exemplarynucleic acid sequence which may be used in accordance with the presentinvention may be purchased from any manufacturer, as for example, fromGenecopoeia (miArrest, microRNA vector based inhibitors).

Thus, according to another embodiment, there is provide an isolatedpolynucleotide comprising a nucleic acid sequence for downregulating anexpression of miR-181, miR-182, miR-26, miR-27, miR-135, miR-335, miR-15and miR-19 or a precursor thereof.

Exemplary polynucleotides which may be used in accordance with thepresent invention to downregulate the expression of miR-181 include, butare not limited to, those set in SEQ ID NOs: 134-137 and SEQ ID NOs:154-157.

Exemplary polynucleotides which may be used in accordance with thepresent invention to downregulate the expression of miR-182 include, butare not limited to, those set in SEQ ID NOs: 138-141 and SEQ ID NO: 147.

Exemplary polynucleotides which may be used in accordance with thepresent invention to downregulate the expression of miR-26 include, butare not limited to, those set in SEQ ID NOs: 126-129 and SEQ ID NOs:145-146.

Exemplary polynucleotides which may be used in accordance with thepresent invention to downregulate the expression of miR-27 include, butare not limited to, those set in SEQ ID NOs: 130-133 and SEQ ID NOs:152-153.

Exemplary polynucleotides which may be used in accordance with thepresent invention to downregulate the expression of miR-135 include, butare not limited to, those set in SEQ ID NOs: 110-113 and SEQ ID NOs:142-143.

Exemplary polynucleotides which may be used in accordance with thepresent invention to downregulate the expression of miR-335 include, butare not limited to, those set in SEQ ID NOs: 114-117 and SEQ ID NO: 144.

Exemplary polynucleotides which may be used in accordance with thepresent invention to downregulate the expression of miR-15 include, butare not limited to, those set in SEQ ID NOs: 118-121 and SEQ ID NOs:150-151.

Exemplary polynucleotides which may be used in accordance with thepresent invention to downregulate the expression of miR-19 include, butare not limited to, those set in SEQ ID NOs: 122-125 and SEQ ID NOs:148-149.

Such nucleic acid sequences may be further comprised in an expressionvector as described in further detail hereinabove.

The present invention further contemplates assessing the expression ofthe target gene (e.g. transcript or polypeptide) followingdownregulating or upregulating the microRNA level in the cell (e.g.neuroglia cell or cardiac cell).

Thus, the presence and/or level of a target gene (e.g. Slc6a4, Htr1a,MaoA, Adrb1, Adrb2,_CRH type 1 receptor, CB1, FKBP5, Tph2, Grm1, Grik3,Grm5, Grik2, Grm7, Gria2, Dscam, L1cam, Tsnax, Sgk1 and/or Stx1a)nucleic acid sequence (e.g. transcript) can be determined using anisolated polynucleotide (e.g., a polynucleotide probe, anoligonucleotide probe/primer) capable of hybridizing to a target gene'snucleic acid sequence (e.g. Slc6a4 as set forth in e.g. NM_001045.4 or aportion thereof; Htr1a as set forth in e.g. NM_000524.3 or a portionthereof; MaoA as set forth in e.g. NM_000240.3 or NM_001270458.1 or aportion thereof; Adrb1 as set forth in e.g. NM_000684.2 or a portionthereof; Adrb2 as set forth in e.g. NM_000024.5 or a portion thereof;CRH type 1 receptor as set forth in e.g. NM_001145146.1, NM_001145147.1or a portion thereof; CB1 as set forth in e.g. NM_001160226.1,NM_033181.3 or a portion thereof; FKBP5 as set forth in e.g.NM_001145775.1, NM_001145777.1 or a portion thereof; Tph2 as set forthin e.g. NM_173353.3 or a portion thereof; Grm1 as set forth in e.g.NM_000838.3, NM_001114329.1 or a portion thereof; Grik3 as set forth ine.g. NM_000831.3 or a portion thereof; Grm5 as set forth in e.g.NM_000842.3, NM_001143831.2 or a portion thereof; Grik2 as set forth ine.g. NM_001166247.1, NM_021956.4 or a portion thereof; Grm7 as set forthin e.g. NM_000844.3, NM_181874.2 or a portion thereof; Gria2 as setforth in e.g. NM_000826.3, NM_001083619.1 or a portion thereof; Dscam asset forth in e.g. NM_001389.3 or a portion thereof; L1cam as set forthin e.g. NM_000425.3, NM_001143963.1, NM_024003.2 or a portion thereof;Tsnax_as set forth in e.g. NM_005999.2 or a portion thereof; Sgk1 as setforth in e.g. NM_001143676.1, NM_001143677.1, NM_001143678.1 or aportion thereof and/or Stx1a as set forth in e.g. NM_001165903.1,NM_004603.3 or a portion thereof). Such a polynucleotide can be at anysize, such as a short polynucleotide (e.g., of 15-200 bases), andintermediate polynucleotide (e.g., 200-2000 bases) or a longpolynucleotide larger of 2000 bases.

The isolated polynucleotide probe used by the present invention can beany directly or indirectly labeled RNA molecule (e.g., RNAoligonucleotide, an in vitro transcribed RNA molecule), DNA molecule(e.g., oligonucleotide, cDNA molecule, genomic molecule) and/or ananalogue thereof [e.g., peptide nucleic acid (PNA)] which is specific tothe target gene RNA transcript of the present invention.

Oligonucleotides designed according to the teachings of the presentinvention can be generated according to any oligonucleotide synthesismethod known in the art, as described in detail hereinabove.

The oligonucleotide of the present invention is of at least 17, at least18, at least 19, at least 20, at least 22, at least 25, at least 30 orat least 40, bases specifically hybridizable with sequence alterationsdescribed hereinabove.

The oligonucleotides of the present invention may comprise heterocylicnucleosides consisting of purines and the pyrimidines bases, bonded in a3′ to 5′ phosphodiester linkage.

Preferably used oligonucleotides are those modified in either backbone,internucleoside linkages or bases, as is broadly described hereinabove.

The isolated polynucleotide used by the present invention can be labeledeither directly or indirectly using a tag or label molecule. Such labelscan be, for example, fluorescent molecules (e.g., fluorescein or TexasRed), radioactive molecule (e.g., ³²P-γ-ATP or ³²P-α-ATP) andchromogenic substrates [e.g., Fast Red, BCIP/INT, available from (ABCAM,Cambridge, Mass.)]. Direct labeling can be achieved by covalentlyconjugating a label molecule to the polynucleotide (e.g., usingsolid-phase synthesis) or by incorporation via polymerization (e.g.,using an in vitro transcription reaction or random-primed labeling).Indirect labeling can be achieved by covalently conjugating orincorporating to the polynucleotide a non-labeled tag molecule (e.g.,Digoxigenin or biotin) and subsequently subjecting the polynucleotide toa labeled molecule (e.g., anti-Digoxigenin antibody or streptavidin)capable of specifically recognizing the non-labeled tag.

The above-described polynucleotides can be employed in a variety of RNAdetection methods such as Northern blot analysis, reverse-transcribedPCR (RT-PCR) [e.g., a semi-quantitative RT-PCR, quantitative RT-PCRusing e.g., the Light Cycler™ (Roche)], RNA in situ hybridization(RNA-ISH), in situ RT-PCR stain [e.g., as described in Nuovo G J, et al.1993, Intracellular localization of polymerase chain reaction(PCR)-amplified hepatitis C cDNA. Am J Surg Pathol. 17: 683-90, andKomminoth P, et al. 1994, Evaluation of methods for hepatitis C virusdetection in archival liver biopsies. Comparison of histology,immunohistochemistry, in situ hybridization, reverse transcriptasepolymerase chain reaction (RT-PCR) and in situ RT-PCR. Pathol ResPract., 190: 1017-25] and oligonucleotide microarray analysis [e.g.,using the Affymetrix microarray (Affymetrix®, Santa Clara, Calif.)].

The presence and/or level of the target gene (e.g. Slc6a4, Htr1a, MaoA,Adrb1, Adrb2, CRH type 1 receptor, CB1, FKBP5, Tph2, Grm1, Grik3, Grm5,Grik2, Grm7, Gria2, Dscam, L1cam, Tsnax, Sgk1 and/or Stx1a) amino acidsequence (e.g. protein) can be determined using, for example, a specificantibody via the formation of an immunocomplex [i.e., a complex formedbetween the target gene antigen (an amino acid sequence) present in thebiological sample and the specific antibody].

The immunocomplex of the present invention can be formed at a variety oftemperatures, salt concentration and pH values which may vary dependingon the method and the biological sample used and those of skills in theart are capable of adjusting the conditions suitable for the formationof each immunocomplex.

The term “antibody” as used in this invention includes intact moleculesas well as functional fragments thereof, such as Fab, F(ab′)2, Fv orsingle domain molecules such as VH and VL to an epitope of an antigen.These functional antibody fragments are defined as follows: (1) Fab, thefragment which contains a monovalent antigen-binding fragment of anantibody molecule, can be produced by digestion of whole antibody withthe enzyme papain to yield an intact light chain and a portion of oneheavy chain; (2) Fab′, the fragment of an antibody molecule that can beobtained by treating whole antibody with pepsin, followed by reduction,to yield an intact light chain and a portion of the heavy chain; twoFab′ fragments are obtained per antibody molecule; (3) (Fab′)2, thefragment of the antibody that can be obtained by treating whole antibodywith the enzyme pepsin without subsequent reduction; F(ab′)2 is a dimerof two Fab′ fragments held together by two disulfide bonds; (4) Fv,defined as a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; (5) Single chain antibody (“SCA”), agenetically engineered molecule containing the variable region of thelight chain and the variable region of the heavy chain, linked by asuitable polypeptide linker as a genetically fused single chainmolecule; and (6) Single domain antibodies are composed of a single VHor VL domains which exhibit sufficient affinity to the antigen.

Methods of producing polyclonal and monoclonal antibodies as well asfragments thereof are well known in the art (See for example, Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,New York, 1988, incorporated herein by reference).

Antibody fragments according to the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ormammalian cells (e.g. Chinese hamster ovary cell culture or otherprotein expression systems) of DNA encoding the fragment. Antibodyfragments can be obtained by pepsin or papain digestion of wholeantibodies by conventional methods. For example, antibody fragments canbe produced by enzymatic cleavage of antibodies with pepsin to provide a5S fragment denoted F(ab′)2. This fragment can be further cleaved usinga thiol reducing agent, and optionally a blocking group for thesulfhydryl groups resulting from cleavage of disulfide linkages, toproduce 3.5S Fab′ monovalent fragments. Alternatively, an enzymaticcleavage using pepsin produces two monovalent Fab′ fragments and an Fcfragment directly. These methods are described, for example, byGoldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and referencescontained therein, which patents are hereby incorporated by reference intheir entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)].Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Fv fragments comprise an association of VH and VL chains. Thisassociation may be noncovalent, as described in Inbar et al. [Proc.Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variablechains can be linked by an intermolecular disulfide bond or cross-linkedby chemicals such as glutaraldehyde. Preferably, the Fv fragmentscomprise VH and VL chains connected by a peptide linker. Thesesingle-chain antigen binding proteins (scFv) are prepared byconstructing a structural gene comprising DNA sequences encoding the VHand VL domains connected by an oligonucleotide. The structural gene isinserted into an expression vector, which is subsequently introducedinto a host cell such as E. coli. The recombinant host cells synthesizea single polypeptide chain with a linker peptide bridging the two Vdomains. Methods for producing scFvs are described, for example, byWhitlow and Filpula, Methods 2: 97-105 (1991); Bird et al., Science242:423-426 (1988); Pack et al., Bio/Technology 11:1271-77 (1993); andU.S. Pat. No. 4,946,778, which is hereby incorporated by reference inits entirety.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick and Fry[Methods, 2: 106-10 (1991)].

Antibodies can also be produced using various techniques known in theart, including phage display libraries [Hoogenboom and Winter, J. Mol.Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. Thetechniques of Cole et al. and Boerner et al. are also available for thepreparation of human monoclonal antibodies (Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner etal., J. Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies canbe made by introduction of human immunoglobulin loci into transgenicanimals, e.g., mice in which the endogenous immunoglobulin genes havebeen partially or completely inactivated. Upon challenge, human antibodyproduction is observed, which closely resembles that seen in humans inall respects, including gene rearrangement, assembly, and antibodyrepertoire. This approach is described, for example, in U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and inthe following scientific publications: Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison,Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14,845-51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); andLonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).

Exemplary antibodies which may be used in accordance with the presentinvention include e.g. anti-Slc6a4 antibody available e.g. from AbnovaCorporation, Abgent and MBL International; anti-Htr1a antibody availablee.g. from Novus Biologicals, Acris Antibodies GmbH and AbnovaCorporation; anti-MaoA antibody available e.g. from Abnova Corporation,Proteintech Group, Inc. and Abgent; anti-Adrb1 antibody available e.g.from Biorbyt, Abgent and antibodies-online; anti-Adrb2 antibodyavailable e.g. from Tocris Bioscience, Abnova Corporation andantibodies-online; anti-CRH type 1 receptor antibody available e.g. fromMyBioSource(dot)com, Abcam and Novus Biologicals; anti-CB1 antibodyavailable e.g. from Santa Cruz Biotechnology, Inc. and Epitomics, Inc.;anti-FKBP5 antibody available e.g. from BD Biosciences and AbnovaCorporation; anti-Tph2 antibody available e.g. from Novus Biologicalsand Acris Antibodies GmbH; anti-Grm1 antibody available e.g. from NovusBiologicals and Biorbyt; anti-Grik3 antibody available e.g. from AcrisAntibodies GmbH and Atlas Antibodies; anti-Grm5 antibody available e.g.from Biorbyt and Acris Antibodies GmbH; anti-Grik2 antibody availablee.g. from Proteintech Group, Inc., Aviva Systems Biology and Abgent;anti-Grm7 antibody available e.g. from Acris Antibodies GmbH andantibodies-online; anti-Gria2 antibody available e.g. from ProteintechGroup, Inc. and Abnova Corporation; anti-Dscam antibody available e.g.from Novus Biologicals and R&D Systems; anti-L1cam antibody availablee.g. from GeneTex, Novus Biologicals and Acris Antibodies GmbH;anti-Tsnax antibody available e.g. from BD Biosciences and GenWayBiotech, Inc.; anti-Sgk1 antibody available e.g. from Epitomics, Inc.and Acris Antibodies GmbH; and/or anti-Stx1a antibody available e.g.from MBL International and Spring Bioscience.

Various methods can be used to detect the formation of the immunocomplexof the present invention and those of skills in the art are capable ofdetermining which method is suitable for each immunocomplex and/or thetype of cells used for diagnosis.

The specific antibody (e.g. anti-Slc6a4 antibody; anti-Htr1a antibody;anti-MaoA antibody; anti-Adrb1 antibody; anti-Adrb2 antibody; anti-CRHtype 1 receptor antibody; anti-CB1 antibody; anti-FKBP5 antibody;anti-Tph2 antibody; anti-Grm1 antibody; anti-Grik3 antibody; anti-Grm5antibody; anti-Grik2 antibody; anti-Grm7 antibody; anti-Gria2 antibody;anti-Dscam antibody; anti-L1cam antibody; anti-Tsnax antibody; anti-Sgk1antibody and/or anti-Stx1a antibody) used in the immunocomplex of thepresent invention can be labeled using methods known in the art. It willbe appreciated that the labeled antibodies can be either primaryantibodies (i.e., which bind to the specific antigen, e.g., a targetgene-specific antigen) or secondary antibodies (e.g., labeled goat antirabbit antibodies, labeled mouse anti human antibody) which bind to theprimary antibodies. The antibody can be directly conjugated to a labelor can be conjugated to an enzyme.

Antibodies of the present invention can be fluorescently labeled (usinga fluorescent dye conjugated to an antibody), radiolabeled (usingradiolabeled e.g., ¹²⁵I, antibodies), or conjugated to an enzyme (e.g.,horseradish peroxidase or alkaline phosphatase) and used along with achromogenic substrate to produce a colorimetric reaction. Thechromogenic substrates utilized by the enzyme-conjugated antibodies ofthe present invention include, but are not limited to, AEC, Fast red,ELF-97 substrate[2-(5′-chloro-2-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone],p-nitrophenyl phosphate (PNPP), phenolphthalein diphosphate, and ELF39-phosphate, BCIP/INT, Vector Red (VR), salmon and magenta phosphate(Avivi C., et al., 1994, J Histochem. Cytochem. 1994; 42: 551-4) foralkaline phosphatase enzyme and Nova Red, diaminobenzidine (DAB),Vector(R) SG substrate, luminol-based chemiluminescent substrate for theperoxidase enzyme. These enzymatic substrates are commercially availablefrom Sigma (St Louis, Mo., USA), Molecular Probes Inc. (Eugene, Oreg.,USA), Vector Laboratories Inc. (Burlingame, Calif., USA), ZymedLaboratories Inc. (San Francisco, Calif., USA), Dako Cytomation(Denmark).

Detection of the immunocomplex in a biological sample, such as bloodsample or serum, which may contain soluble (e.g., secreted, shedded)target gene polypeptide can be performed using fluorescence activatedcell sorting (FACS), enzyme linked immunosorbent assay (ELISA), Westernblot and radio-immunoassay (RIA) analyses, immunoprecipitation (IP) orby a molecular weight-based approach.

For Western blot the proteins are extracted from a cell sample and aresubjected to electrophoresis (e.g., SDS-PAGE) and blotting to a membrane(e.g., nylon or PVDF). The membrane is then interacted with a specificantibody (e.g. anti-Slc6a4 antibody; anti-Htr1a antibody; anti-MaoAantibody; anti-Adrb1 antibody; anti-Adrb2 antibody; anti-CRH type 1receptor antibody; anti-CB1 antibody; anti-FKBP5 antibody; anti-Tph2antibody; anti-Grm1 antibody; anti-Grik3 antibody; anti-Grm5 antibody;anti-Grik2 antibody; anti-Grm7 antibody; anti-Gria2 antibody; anti-Dscamantibody; anti-L1cam antibody; anti-Tsnax antibody; anti-Sgk1 antibodyand/or anti-Stx1a antibody) which can be either directly labeled orfurther subjected to a secondary labeled antibody. Detection may be byautoradiography, colorimetric reaction or chemiluminescence. This methodallows both quantitation of an amount of substrate and determination ofits identity by a relative position on the membrane which is indicativeof a migration distance in the acrylamide gel during electrophoresis.

In case the concentration of the antigen in the biological sample islow, detection of the antigen (target gene amino acid sequence) can beperformed by immunoprecipitation (IP). For immunoprecipitation analysisthe specific antibody (e.g. anti-Slc6a4 antibody; anti-Htr1a antibody;anti-MaoA antibody; anti-Adrb1 antibody; anti-Adrb2 antibody; anti-CRHtype 1 receptor antibody; anti-CB1 antibody; anti-FKBP5 antibody;anti-Tph2 antibody; anti-Grm1 antibody; anti-Grik3 antibody; anti-Grm5antibody; anti-Grik2 antibody; anti-Grm7 antibody; anti-Gria2 antibody;anti-Dscam antibody; anti-L1cam antibody; anti-Tsnax antibody; anti-Sgk1antibody and/or anti-Stx1a antibody) may directly interact with a sample(e.g., cell lysate) including the target gene polypeptide and the formedcomplex can be further detected using a secondary antibody conjugated tobeads (e.g., if the specific antibody is a mouse monoclonal antibody,the secondary antibody may be an anti-mouse antibody conjugated to e.g.,Sepharose beads). The beads can be then precipitated by centrifugation,following which the precipitated proteins (e.g., target gene polypeptideand specific antibodies) can be detached from the beads (e.g., usingdenaturation at 95° C.) and further subjected to Western blot analysisusing antibodies. Alternatively, the specific antibody and thebeads-conjugated secondary antibody may be added to the biologicalsample containing the antigen (target gene polypeptide) to thereby forman immunocomplex. Alternatively, if the target gene polypeptide is ahighly glycosilated protein, it can be also precipitated using asubstrate capable of binding glycosilated polypeptides such Concavalin A(GE Healthcare Bio-Sciences, Uppsala, Sweden) which may be alsoconjugated to beads, followed by Western blot analysis specificantibodies as described above.

FACS analysis enables the detection of antigens present on cellmembranes. Briefly, specific antibodies, as described above, are linkedto fluorophores and detection is performed by means of a cell sortingmachine which reads the wavelength of light emitted from each cell as itpasses through a light beam. This method may employ two or moreantibodies simultaneously.

The presence and/or level of target gene polypeptide can be alsodetermined using ELISA. Briefly, a sample containing the target geneantigen is fixed to a surface such as a well of a microtiter plate. Anantigen specific antibody (e.g. anti-Slc6a4 antibody; anti-Htr1aantibody; anti-MaoA antibody; anti-Adrb1 antibody; anti-Adrb2 antibody;anti-CRH type 1 receptor antibody; anti-CB1 antibody; anti-FKBP5antibody; anti-Tph2 antibody; anti-Grm1 antibody; anti-Grik3 antibody;anti-Grm5 antibody; anti-Grik2 antibody; anti-Grm7 antibody; anti-Gria2antibody; anti-Dscam antibody; anti-L1cam antibody; anti-Tsnax antibody;anti-Sgk1 antibody and/or anti-Stx1a antibody) coupled to an enzyme isapplied and allowed to bind to the antigen. Presence of the antibody isthen detected and quantitated by a colorimetric reaction employing theenzyme coupled to the antibody. Enzymes commonly employed in this methodinclude horseradish peroxidase and alkaline phosphatase. If wellcalibrated and within the linear range of response, the amount ofsubstrate present in the sample is proportional to the amount of colorproduced. A substrate standard is generally employed to improvequantitative accuracy.

The presence and/or level of a target gene polypeptide can be alsodetermined using radio-immunoassay (RIA). In one version, this methodinvolves precipitation of the desired antigen (target gene polypeptide)with a specific antibody and radiolabeled antibody binding protein(e.g., protein A labeled with I¹²⁵) immobilized on a precipitablecarrier such as agarose beads. The number of counts in the precipitatedpellet is proportional to the amount of antigen.

In an alternate version of the RIA, a labeled antigen and an unlabelledantibody binding protein are employed. A sample containing an unknownamount of antigen is added in varying amounts. The decrease inprecipitated counts from the labeled antigen is proportional to theamount of antigen in the added sample.

The presence and/or level of a target gene polypeptide can be alsodetermined using molecular weight-based approach. Since theimmunocomplex exhibits a higher molecular weight than its components,methods capable of detecting such a change in the molecular weight canbe also employed. For example, the immunocomplex can be detected by agel retardation assay. Briefly, a non-denaturing acrylamide gel isloaded with samples. A shift in the size (molecular weight) of theprotein product as compared with its components is indicative of thepresence of an immunocomplex. Such a shift to a higher molecular weightcan be viewed using a non-specific protein staining such as silver stainor Commassie blue stain.

In situ detection of the target gene polypeptide in a biological samplesuch as a tissue section (e.g., paraffin embedded or cryosection) can beperformed using immunological staining methods which detects the bindingof antibodies on the cells in situ. Examples of immunological stainingprocedures include but are not limited to, fluorescently labeledimmunohistochemistry (using a fluorescent dye conjugated to anantibody), radiolabeled immunohistochemistry (using radiolabeled e.g.,¹²⁵I, antibodies), and immunocytochemistry [using an enzyme (e.g.,horseradish peroxidase or alkaline phosphatase) and a chromogenicsubstrate to produce a colorimetric reaction]. It will be appreciatedthat the enzymes conjugated to antibodies can utilize variouschromogenic substrates as described hereinabove.

Preferably, the immunological staining used by the present invention isimmunohistochemistry and/or immunocytochemistry.

Immunological staining is preferably followed by counterstaining thecells using a dye, which binds to non-stained cell compartments. Forexample, if the labeled antibody binds to antigens present on the cellcytoplasm, a nuclear stain (e.g., Hematoxylin-Eosin stain) is anappropriate counterstaining.

According to one embodiment, the method comprises measuring anexpression of the Tph2 gene following the downregulating of the miR-181and/or the miR-27.

According to one embodiment, the method comprises measuring anexpression of the Slc6a4 gene following upregulating the miR-135 and/ormiR-335.

According to one embodiment, the method comprises measuring anexpression of the Htr1a gene following upregulating the miR-135,miR-335, miR-181, miR-182 and/or miR-26.

According to one embodiment, the method comprises measuring anexpression of the MaoA gene following upregulating the upregulating themiR-27.

According to one embodiment, the method comprises measuring anexpression of the Adrb1 gene following upregulating the miR-19.

According to one embodiment, the method comprises measuring anexpression of the CB1 gene following upregulating the CB1.

According to one embodiment, the method comprises measuring anexpression of the CRH type 1 receptor gene following upregulating themiR-15.

According to one embodiment, the method comprises measuring anexpression of the FKBP5 gene following upregulating the miR-15.

The present inventors have further realized that mR135 is upregulated insubjects having a serotonin-associated medical condition (describedabove).

Thus, there is provided a method of diagnosing a serotonin-relatedmedical condition in a subject in need thereof, the method comprisingmeasuring an expression level of a miR-135 in a blood of the subject,wherein a high expression level of the miR-135 as compared to that in ablood sample of a healthy subject is indicative of theserotonin-associated medical condition.

Methods of analyzing miR in blood samples are well known in the art andare described hereinbelow.

Diagnosis can be further assessed and established using Gold-standardmethods. Typically, at least one of a full patient medical history,physical assessment, and thorough evaluation of symptoms helps determinethe cause of the depression. Standardized questionnaires can be helpfulsuch as the Hamilton Rating Scale for Depression, and the BeckDepression Inventory.

The present inventors have further shown that miR-135a plasma levels aredecreased in subjects treated with an anti-depressant drug, such asFluoxetine (an anti-depressant of the SSRI class), while brain miR-135alevels are increased in these same subjects (see FIGS. 3E-3J).

Thus, according to another embodiment of the present invention, there isprovided a method of monitoring treatment of an anti-depressant drug,the method comprising: (a) treating a subject in need thereof with ananti-depressant drug; and (b) measuring an expression level of a miR-135in the blood of the subject prior to and following the treatment,wherein a lower expression level of the miR-135 following to thetreatment by the anti-depressant drug as compared to the expressionlevel of the miR-135 prior to the treatment by the anti-depressant drugis indicative of an efficient treatment.

As used herein, the term “anti-depressant drug” refers to any medicationused to alleviate mood disorders, such as major depression anddysthymia, and anxiety disorders, such as social anxiety disorder.Exemplary anti-depressant drugs include, but are not limited to,Selective serotonin reuptake inhibitors (SSRIs, such as Citalopram,Escitalopram, Fluoxetine, Fluvoxamine, Paroxetine and Sertraline);Serotonin-norepinephrine reuptake inhibitors (SNRIs, such asDesvenlafaxine, Duloxetine, Milnacipran and Venlafaxine); Noradrenergicand specific serotonergic antidepressants (such as Mianserin andMirtazapine); Norepinephrine (noradrenaline) reuptake inhibitors (NRIs,such as Atomoxetine, Mazindol, Reboxetine and Viloxazine);Norepinephrine-dopamine reuptake inhibitors (such as Bupropion);Selective serotonin reuptake enhancers (such as Tianeptine);Norepinephrine-dopamine disinhibitors (NDDIs such as Agomelatine);Tricyclic antidepressants (including Tertiary amine tricyclicantidepressants and Secondary amine tricyclic antidepressants); andMonoamine oxidase inhibitor (MAOIs).

According to a specific embodiment, the anti-depressant drug comprisesselective serotonin reuptake inhibitors (SSRI) or noradrenaline reuptakeinhibitors (NRI).

Measuring the expression level of miR-135 is typically effected in ablood sample obtained from the subject.

As used herein, the term “blood sample” refers to fresh whole blood,fractionated whole blood and blood plasma. The blood sample is typicallyobtained from the subject following to treatment with an anti-depressantdrug, however, a blood sample may also be obtained from the subjectprior to treatment for further comparison of miR-135 levels.

An efficient anti-depressant treatment is determined when a lowerexpression level of the miR-135 is obtained following to the treatmentas compared to the miR-135 expression level prior to the treatment.

According to another embodiment, there is provided a method ofmonitoring a psychiatric condition in a subject in need thereof, themethod comprising measuring an expression level of a miR-135 in a bloodof the subject, wherein a high expression level of the miR-135 ascompared to a healthy subject is indicative of the psychiatriccondition.

According to another embodiment, the psychiatric condition comprises adepression, an anxiety, a stress, a fatigue, an impaired cognitivefunction, a panic attack, a compulsive behavior, an addiction, a socialphobia, a sleep disorder and a food related disorder.

According to a specific embodiment, miR-135 comprises miR-135a.

Measuring an expression level of a miR-135 may be carried out by anymethod known to one of ordinary skill in the art, as for example, bynorthern analysis, RNase protection assay, and PCR (e.g. real-time PCR).

Monitoring treatment may also be effected by assessing the patient'swell being, and additionally or alternatively, by subjecting the subjectto behavioral tests, MRI or any other method known to one of skill inthe art.

It is expected that during the life of a patent maturing from thisapplication many relevant inhibitors of miRNAs or alternatively miRNAmodifications will be developed and the scope of the term microRNAs isintended to include all such new technologies a priori.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader.

All the information contained therein is incorporated herein byreference.

Example 1 Differential Expression of miRs in Serotonin Neurons

Materials and Experimental Procedures

5HT Neurons MicroRNA Microarray

Hindbrain cells from embryonic day 12 of ePET YFP mice were cultured andsorted to distinguish 5HT neurons from surrounding non-5HT neurons.Total RNA including the miRNA population was purified, labeled andhybridized on Agilent Mouse miRNA Microarray (Agilent Tech, Mississauga,ON, Canada) design number 021828 based on Sanger miRBase release 12.0according to manufactures instructions. The microarrays were scanned andthe data was extracted and processed using the Feature ExtractionSoftware (Agilent Technologies). Following scanning, intensity outputdata of the GeneView.txt files was analyzed to quantify differentialrelative expression of microRNAs using the Partek® Genomics Suite(Partek Inc., St. Louis, Mo.). The data was log 2 transformed, quantilenormalized and filtered according to the flag “gIsGeneDetected” in theGeneView file. Of 666 murine miRs, 198 remained for further analysisupon this filtering step. Differentially expressed miRs were thenidentified by using a threshold of a 1.5 fold change with significanceaccording to ANOVA. Contrasts were calculated within the ANOVA test. TheBenjamini and Hochberg correction was used for false-positive reduction(multiple testing correction).

Cloning of 3′ UTRs into Psicheck2 Luciferase Expression Plasmid

3′UTR sequences of Slc6a4, Htr1a, MaoA and Tph2 were PCR amplified frommouse genomic DNA, or total brain cDNA. 3′UTR PCR fragments were ligatedinto pGEM-T easy vector (Promega) according to the manufacturer'sguidelines, and further subcloned into a single NotI site at the 3′ endof luciferase in the Psicheck2 reporter plasmid (Promega). Mutated 3′UTR sequences, lacking miR-135 seed sequences, were synthesized withprimers overhangs across the seed match sequence. Cloning orientationwas verified by diagnostic cuts and by sequencing.

Transfections and Luciferase Assay

HEK293T cells were grown on poly-L-lysine in 48-well format to a 70-85%confluence and transfected using Polyethyleneimine with the followingplasmids: 5 ng of Psicheck2-3′UTR plasmid and 215 ng of over-expressingvector for a specific miRNA, or empty-miR-vec over-expression plasmids.24 hours following transfection cells were lysed and luciferasereporters activity were assayed as previously described [Chen A. et al.Mol Endocrinol (2005) 19: 441-58]. Renilla luciferase values werenormalized to control firefly luciferase levels (transcribed from thesame vector but not affected by 3′UTR tested) and averaged across sixwell repetitions per condition.

Animals and Housing

Adult C57BL/6J male mice, 10 weeks old (Harlan, Jerusalem, Israel) werehoused in a temperature-controlled room (22±1° C.) on a reverse 12 hourlight/dark cycle. Food and water were available ad libitum. Allexperimental protocols were approved by the Institutional Animal Careand Use Committee of The Weizmann Institute of Science.

Acute Immobilization Stress Paradigms

Adult mice were introduced into a 50 ml ventilated tube for 30 minutesduring their dark cycle.

Chronic Social Defeat

Mice were subjected to a social defeat protocol as previously described[Krishnan V. et al. Cell (2007) 131: 391-404]. Briefly, the mice wereplaced in a home cage of an aggressive ICR mouse and they physicallyinteracted for five minutes. During this time, the ICR mouse attackedthe intruder mouse and the intruder displayed subordinate posturing. Aperforated clear plexiglass dividers were then placed between theanimals and the mice remained in the same cage for 24 hours to allowsensory contact. The procedure was then repeated with an unfamiliar ICRmouse for each of the next 10 days.

Antidepressant Treatment

Mice received i.p. injection of tricyclic-Imipramine, orSSRI-Fluoxetine, or NRI-Reboxetine (20 mg/kg in saline) or saline.Chronic injections were carried out for 18-21 consecutive days, and anacute injection was performed 24 hours prior to brain microdissections.

Microdissection of the Raphe Nucleus and Plasma Collections

Brain samples were taken from mice raphe nucleus (RN) after removing thebrain and placing it on acryl brain matrix (Stoelting). Slices weretaken using standard razor blades (GEM) based on designated anatomicalmarkers. Blunted 14G syringes were used to extract the RN region from 3mm slices removed from the matrix. Additionally, trunk blood wascollected in EDTA containing tubes to avoid coagulation. Aftercentrifugation in 3,500 g for 30 minutes at 4° C., plasma was separatedand kept at −70° C. until RNA purification.

microRNA Purification and Quantitative RT-PCR Expression Analysis

mRNAs, including microRNAs, were isolated from sorted neurons, frozenbrain punches and plasma using miRNeasy mini kit (Qiagen) according tothe manufacturer instructions, and treated using miScript Reversetranscription kit miRNA to generate cDNA. cDNA samples were thenanalyzed using SYBR®Green PCR kit (Qiagen) according to themanufacturer's guidelines in AB 7500 thermocycler (Applied Biosystems).Specific primers for each miR were used together with the commercialuniversal primer, while U6 snRNA was used as internal control.

TABLE 1B Primers sequences used for real time PCR SEQ ID NO.Primer sequence Gene 1 TATGGCTTTTTATTCCTATGTGA miR135a 2TATGGCTTTTCATTCCTATGTGA miR135b 3 TTTGTTCGTTCGGCTCGCGTGA miR375 4GATGACACGCAAATTCGTGAA U6 5 TAAGGCACGCGGTGAATGCC miR124

TABLE 1C Primers sequences used for molecular cloning ProductPrimer Sequence Orientation size Gene AGTTCTGCCGCTGATGATG sense2600 ^(with 2) Htr1a 3′ UTR  1 (SEQ ID NO: 6) GCACAAATGGAGAGTCTGATTantisense Htr1a 3′ UTR  2 AAA (SEQ ID NO: 7) TGCCTTTAATGCAAAACAGC sense2000 ^(with 4) MaoA 3′UTR  3 (SEQ ID NO: 8) CCAAGTTTACAACCATCAAGCantisense MaoA 3′UTR  4 A (SEQ ID NO: 9) ATCCGCATGAATGCTGTGTA sense 760 ^(with 6) Slc6a4 3′UTR  5 (SEQ ID NO: 10) GTGGGTGGTGGAAGAGACACantisense Slc6a4 3′UTR  6 (SEQ ID NO: 11) CCTACACGCAGAGCATTGAA sense 870 ^(with 8) Tph2 3′ UTR  7 (SEQ ID NO: 12) ACATCCCTGTGGGATTTGAGantisense Tph2 3′ UTR  8 (SEQ ID NO: 13) TGTCTTGCTTATATTTTCTCAGT sense 320 ^(with 6) Slc6a4 3′UTR  9 AG (SEQ ID NO: 14) mutatedGAAAATATAAGCAAGACATCC antisense  440 ^(with 5) Slc6a4 3′UTR 10CTGTT (SEQ ID NO: 15) mutated AAAGATCCCTTTCCCCAATG sense 1400 ^(with 12)Htr1a 3′ UTR 11 (SEQ ID NO: 16) short CAGTGCGTCTTCTCCACAGA antisenseHtr1a 3′ UTR 12 (SEQ ID NO: 17) short ATAAGCAAGGGCCCAAAAGGA sense1300 ^(with 12) Htr1a 3′ UTR 13 AGA (SEQ ID NO: 18) mutated seed 1TTTTGGGCCCTTGCTTATAAGT antisense  120 ^(with 11) Htr1a 3′ UTR 14CC (SEQ ID NO: 19) mutated seed 1 CTGCCCTGCCACATGTGTTTTT sense 170 ^(with 12) Htr1a 3′ UTR 15 AT (SEQ ID NO: 20) mutated seed 2TAACAAATAAAAACACATGTG antisense 1260 ^(with 11) Htr1a 3′ UTR 16GCA (SEQ ID NO: 21) mutated seed 2 ACCGGTCATATGATTCCCCAGT sense 199 ^(with 18) Pre-mmu- 17 TTCCTGCTTT (SEQ ID NO: 22) miR135bACCGGTCCTCTGTGGCTGGTCC antisense Pre-mmu- 18 TTAG (SEQ ID NO: 23)miR135b

Cloning of miR135b Over Expression Viral Vector Pre-miR-135b wasamplified by PCR from mouse genomic DNA with primers adding restrictionenzyme AgeI sites and then was inSlc6a4ed to pGEM-T Easy vector(Promega, Madison, Wis.). After sequencing of pGEM-T Easy and digestionof both pGEM-T Easy and pEGFP vector (Clontech laboratories Inc.,Mountain View, Calif.) with the AgeI, the premature miR-135b sequencewas ligated to the pEGFP vector to construct the expression plasmidpEGFP-miR-135b. Afterwards, pEGFP-miR-135b was cut by BamHI and BsrGI inparallel to cutting pCSC-E/Syn-eGFP plasmid with the same enzymes, andthe miR-135b-eGFP sequence was ligated to pCSC-E/Syn to constructpCSC-eSNY-pre-miR-135b-eGFP plasmid which was confirmed by restrictionendonuclease analysis and DNA sequencing.

Production of Lentiviral Vectors

Recombinant lentiviruses were produced by transient transfection inHEK293T cells, as previously described [Naldini L et al., Proc Natl AcadSci USA (1996) 93:11382-8]. Briefly, infectious lentiviruses wereharvested at 48 and 72 hours post-transfection, filtered through 0.45pin-pore cellulose acetate filters and concentrated byultracentrifugation.

Intracerebral Injections of Lentiviruses

To provide precision control over the stereotaxic surgery and site oflentiviral delivery, inventors used a computer-guided stereotaxicinstrument and a motorized nanoinjector (Angle Two™ StereotaxicInstrument, myNeurolab). As previously described [Singer 0. et al. NatNeurosci (2005).8, 1343-9] mice were placed on a stereotaxic apparatusunder general anesthesia, and coordinates were determined as defined bythe Franklin and Paxinos atlas. The lentiviral preparation was deliveredusing a Hamilton syringe connected to the motorized nanoinjector systemand solution injected at a rate of 0.2 μl every 1 min. Following twoweeks recovery period, mice were subjected to behavioral andphysiological studies and afterwards anesthetized and perfused withphosphate buffered 4% paraformaldehyde. The fixed brains were seriallysectioned to 30μ slices in order to confirm the preciseness of theinjection site, using immunohistochemistry.

Immunohistochemistry

The procedure used for immunohistochemistry was carried out aspreviously described [Chen A et al. J Neurosci (2006) 26: 5500-10]. ForGFP immunostaining, inventors used biotinylated anti GFP antibody raisedin rabbit as primary antibody (Abcam, Cambridge, UK), and streptavidinconjugated Cy2 as secondary antibody (Jackson ImmunoresearchLaboratories Inc, West Grove, Pa., USA).

Behavioral Assessments

All behavioral assessments were performed during the dark phasefollowing habituation to the test room for 2 hours prior each test.

Tail Suspension Test

The tail suspension test was performed in the TSE Tail SuspensionMonitor (TSE Systems, Bad Homburg, Germany). Each mouse was taped by thetip of its tail, and suspended from the force sensor for 10 minutes.Time spent immobile and time spent struggling were calculated andrecorded by the software based on pre-set thresholds.

Modified Forced Swim Test

The tail suspension test was performed as previously described [KrishnanV and Nestler E J, Nature (2008) 455: 894-902]. In short, the apparatusused was a plastic bucket, 18 cm of diameter, filled with 25° C. waterto a depth of 15 cm. Each mouse was placed in the center of the bucketto initiate a 6 minutes video recorded test session. The duration oftime spent immobile during the 2-6 minute of testing was automaticallyscored using EtoVision XT (Noldus, Wageningen, Netherlands).

Locomotor Activity

To control for the possibility of behavioral effects originating fromdifferences in ambulatory movement, locomotor activity of mice wasexamined over a 48 hours period, which proceeded a few days ofhabituation. Mice were single housed in specialized home cages andlocomotion was measured using the InfraMot system (TSE Systems, BadHamburg, Germany).

Statistical Analysis

Data were expressed as means+/−SEM. To test for statisticalsignificance, student's t test was used in cases where only two groupswere compared, such as between microarray validation qPCR. One wayANOVAs was used to compare between multiple groups such as between thedifferent treatments in the luciferase assay. Two way ANOVAs was used inthe cases of 2 independent variable, such as the SSRI NRI injection,both in acute and chronic durations. Post hoc t tests were used whennecessary to reveal statistical significance. Differences between groupswere considered significant when P<0.05.

Results

5HT neurons were isolated from the RN of ePET YFP embryos, and their miRexpression profile was compared to non-5HT neurons, obtained from thesame nucleus, using miR microarray (FIG. 1A). Fourteen miRs were foundto be upregulated and twenty-seven downregulated by more than 2 fold in5HT neurons compared to the non-5HT neurons (see Tables 2A-B, below).Representative validation of array results was performed using real timePCR for miRs upregulated in 5HT neurons such as miR-375 (P=0.0071; FIG.1B) and downregulated such as miR-135a (P=0.0075; FIG. 1C). In order tofurther study the role of miRs as modulators of 5HT neurons, extensivebioinformatic analysis was performed in a hypothesis driven manner.Targeting prediction of known serotonin related genes that have beenpreviously demonstrated to be associated with psychopathologies, werecrossed with the microarray results. The following four protein codingtarget genes expressed in 5HT neurons in the RN were chosen for testing:serotonin transporter, responsible for 5HT reuptake (also known as SERTor Slc6a4); serotonin inhibitory receptor 1a (also known as Htr1a);tryptophan hydroxylase 2 (Tph2), the rate limiting enzyme of 5HTsynthesis in the brain; and monoamine hydroxylase (MaoA), whichdeactivates 5HT. MicroRNA targeting predictions for these genes wasperformed using two different web-based algorithms: Target Scan[www(dot)targetscan(dot)org] and Miranda [www(dot)microrna(dot)org] andwere crossed with the list of 91 miRs altered by at least ±1.5 in the5HT neurons miRs array, compared to non-5RH cells. Based on the miRsarray data and the bioinformatic analysis, eight miRs were chosen forfurther in vitro studies (FIG. 1D-1G).

TABLE 2A List of miRs upregulated in 5HT neurons compared tonon-serotonergic (by more than 2 fold). Fold change microRNA name 20.72mmu-miR-375 11.73 mmu-miR-376c 4.44 mmu-miR-7a 2.87 mmu-miR-137 2.79mghv-miR-M1-2 2.61 mmu-miR-709 2.51 mmu-miR-291b-5p 2.40 mmu-miR-12242.37 mmu-miR-1892 2.31 mmu-miR-702 2.25 mmu-miR-139-3p 2.24 mmu-miR-7622.10 mmu-miR-671-5p 2.04 mmu-miR-483*

TABLE 2B List of miRs downregulated in 5HT neurons compared to non-serotonergic (by more than 2 fold). Fold change microRNA name −5.10mmu-miR-691 −4.11 mmu-miR-4661 −3.95 mmu-miR-17 −3.18 mmu-miR-376b −3.13mmu-miR-124 −3.08 mmu-miR-218 −2.99 mmu-miR-128 −2.92 mmu-miR-140* −2.86mmu-miR-148a −2.86 mmu-miR-340-5p −2.82 mmu-miR-181c −2.72 mmu-miR-210−2.69 mmu-miR-135a −2.66 mmu-miR-27a −2.45 mmu-miR-452 −2.20 mmu-miR-370−2.19 mmu-miR-300 −2.17 mmu-miR-376a −2.13 mmu-miR-127 −2.12 mmu-miR-15b−2.07 mmu-miR-101a −2.06 mmu-miR-16 −2.05 mmu-miR-324-5p −2.05mmu-miR-434-5p −2.03 mmu-miR-92a −2.00 mmu-miR-669i

In vitro luciferase assays were performed to test the miR-targetinteractions between the 3′UTR of the tested 5HT related gene and themiRs predicted to putatively target it. Inventors found that Tph2 3′UTRwas mildly repressed (by approximately 20%) by miR-27b (P=0.0051) andmiR-181C (P=0.0305, FIG. 1H) and MaoA 3′UTR was also repressed bymiR-27b (P=0.0008, FIG. 1I). miR-135 targeting of Slc6a4 3′UTR (FIGS. 2Aand 2C) and Htr1a 3′UTR (FIGS. 2B and 2D) resulted in robust repressionof translation of these transcripts. While miR-135a lead toapproximately 30% repression to Slc6a4 (P=0.014) and Htr1a (P<0.0001),miR-135b caused approximately 50% repression to Slc6a4 (P=0.0002) andHtr1a (P<0.0001). Additionally significant repression of Htr1a 3′UTR wasgenerated by miR-335 (P<0.0001), miR-181c (P=0.0029) and miR-26a(P<0.0001, FIG. 2D). Further genomic approach bioinformatic analysisrevealed a strong conservation of miR-135 seed match in the slc6a4 3′UTR(FIG. 2E) and in one out of the two identified seed matches in the Htr1a3′UTR (FIG. 2F). Mutation studies in the 3′UTR of the Slc6a4 transcript,which removed the miR seed match of miR-135, revealed that both miR-135aand miR-135b targeting of Slc6a4 was mediated via its seed matchsequence. The repression induced by the miR-135 was fully blocked by themutation in Slc6a4 3′UTR (FIG. 2G). Mutating the Htr1a miR-135 seedmatches individually or both revealed that miR-135a was repressing Htr1a3′UTR via the distal and not the proximal seed match while miR-135b actvia both predicted sites (FIG. 2H).

Inventors further tested the regulation of RN-miR-135 expression in vivofollowing different environmental challenges or pharmacologicaltreatments. Following manipulation of the mice (i.e. acuteimmobilization stress) RN was removed, RNA was extracted and miR-135levels were tested using real time PCR. Since 5HT levels are known to bealerted by acute stress, inventors tested miR-135 levels in differenttime points after acute restraint stress, and found that both miR-135aand miR-135b were downregulated 90 minutes following acute stress(P<0.0001). The reduced levels of these miRs still remained 24 hoursafter stress, compared to control mice (P=0.0357 for miR-135a, FIG. 3A;P=0.0055 for miR-135b, FIG. 3B). Furthermore, since 5HT neuronalfunctions and Slc6a4 and Htr1a expression levels are known to bestrongly affected in depressed patients and following anti-depressantsmedications, inventors tested the levels of the two miR variants in miceexposed to environmental model for induction of depression-likebehaviors (chronic social defeat model) and to the tricyclicantidepressant, Imipramine. Interestingly, chronic social defeat stressdid not alter miR-135 levels in the raphe nucleus, however, Imipramineadministered acutely or chronically, both in stressed and non stressedmice, increased miR-135a (P=0.003; FIG. 3C) and miR-135b (P=0.0093; FIG.3D) expression levels in the RN. Since Imipramine is not a specific 5HTreuptake inhibitor, inventors further tested the affect of both acuteand chronic selective serotonin reuptake inhibitors (SSRI), Fluoxetine,and the noradrenaline reuptake inhibitors (NRI), Reboxetine, and found arobust increase in miR-135a levels following both acute and chronic SSRItreatment (P<0.0001, FIG. 3E), and not in miR-135b levels in the RN(FIG. 3F). Intrigued by the change in miR-135 levels in the RN followingSSRI treatment, inventors tested the levels of circulating miR-135 inmice plasma, and found a robust decrease in miR-135a levels bothfollowing acute and chronic SSRI administration (main effect for drugP<0.0001, FIG. 3G) and no effect in circulating miR-135b levels (FIG.3H), suggesting a strong reverse correlation between miR-135a levels inthe RN and the plasma following SSRI administration (FIGS. 3I and 3J).

To further explore the importance of miR-135 levels in the whole animalcontext inventors manipulated miR-135 levels in vivo specifically in theRN of adult mice and tested its effects on the mice depression-likebehaviors. To this end, inventors constructed recombinant lentivirusesover-expressing miR-135b specifically in neurons using the enhancedsynapsin promoter, which also co-expressed the GFP reporter (seematerials and experimental procedures section above and FIG. 4A).Inventors tested the lentiviruses in vivo by injecting them into the RNof adult mice, and compared miR-135b levels in RN to controllentiviruses injected mice. Real time PCR analysis of miR-135b levelsrevealed a 10 fold induction compared to control lentiviruses injectedmice (P=0.0032, FIG. 4B). Adult mice injected with miR-135bover-expression were exposed to chronic social defeat, to initiatedepression-like behaviors, and were subsequently tested behaviorally.Following behavioral testing, mice were perfused and brains wereanalyzed for location of injection site (FIGS. 4C-4D). RN miR-135over-expressing mice demonstrated reduced immobility time in the forcedswim (P=0.0088 in minute 3 and P=0.00330 for minute 4; FIG. 4E) and inthe tail suspension tests (P=0.07356 in the last 5 min of the test, FIG.4F) without any observed change in their home cage locomotion (FIGS.4G-4H), suggesting an antidepressant effect for miR-135 over-expression.

Taken together, the present inventors determined the specific miRsexpression fingerprint of the RN serotonergic and non-serotonergicneurons. The present inventors crossed this unique data set withbioinformatics prediction for miRs targeting of 5HT related genes. Thepresent inventors tested in vitro the targeting prediction for Tph2,MaoA, Slc6a4 and Htr1a using 3′UTR's luciferase assays and in mutationstudies and reveled, among other miR-target interactions, a stronginhibitory effect for miR-135 both on Sl6a4 and Htr1a 3′UTR.Furthermore, the inventors demonstrated that miR-135 in the RN isdown-regulated by acute stress, and upregulated by antidepressantadministration, specifically by SSRI drugs. Furthermore, the presentinventors identified a reverse correlation between miR-135a levels inthe RN to its levels in the plasma following SSRI administration.Finally, the present inventors demonstrated that site-specificover-expression of miR-135 in the adult mice RN leads to decreaseddepression-like behaviors following social defeat.

Example 2 miR-19 Specifically Targets Type One Beta Adrenergic Receptor(Adrb1)

Materials and Experimental Procedures

Cloning of 3′ UTRs into Psicheck2 Luciferase Expression Plasmid

3′UTR sequence of ADRb1 was PCR amplified from mouse genomic DNA.Mutated 3′ UTR sequences, lacking all four miR-19 seed matches, wassynthesized by Epoch Biolabs, Inc. (TX, USA). 3′UTR PCR fragments wereligated into pGEM-T easy vector (Promega) according to themanufacturer's guidelines, and further subcloned into a single NotI siteat the 3′ end of luciferase in the Psicheck2 reporter plasmid (Promega).Cloning orientation was verified by diagnostic cuts and by sequencing.

Transfections and Luciferase Assay

HEK293T cells or HT22 neuronal cells were grown on poly-L-lysine in48-well format to a 70-85% confluence and transfected usingPolyethyleneimine with the following plasmids: Psicheck2-3′UTR plasmid,pre-mmu-miR-19b over-expression in pEGFP plasmid or pEGFP plasmid alone(clontech), miR-19b knockdown (KD) plasmid (Genecopoeia) or control-KDplasmid (Genecopoeia). 24 hours following transfection cells were lysedand luciferase reporters activity were assayed as previously described[Chen A. et al. Mol Endocrinol (2005) 19: 441-58]. Renilla luciferasevalues were normalized to control firefly luciferase levels (transcribedfrom the same vector but not affected by 3′UTR tested) and averagedacross six well repetitions per condition.

Results

Bioinformatic analysis for stress related genes with a distinct,evolutionary conserved miRNA target sequences that contain severalrepeats in their 3′UTR revealed miR-19 as a strong candidate for thetargeting of type one beta adrenergic receptor (Adrb1), with threestrongly conserved and one less conserved miR-19 seed match on Adrb13′UTR. Adrb1 is an adrenergic receptor that is expressed in variousregions of the brain including the amygdala, hippocampus andparaventricular nucleus (PVN). Amygdalar Adrb1 was previously describedas affecting anxiety-like behavior [Fu A et al., Brain Res (2008) 1211:85-92; Rudoy C A and Van Bockstaele E J, Prog Neuropsychopharmacol BiolPsychiatry (2007) 31: 1119-29] and fear memory [Roozendaal B et al., JNeurosci (2004) 24: 8161-9; Roozendaal B et al., Neuroscience (2006)138: 901-10]. Intriguingly, Adrb1 was found on CRF positive cells of theamygdala and is a G-protein coupled receptor (GPCR) that exert itseffect via Gs further activating adenylate cyclase (AC). There are 10known genes encoding for AC, namely ADCY1-10. Three of these (ADCY1,ADCY7 and ADCY9) were bioinformatically predicted to be targeted bymiR-19. ADCY1 has a brain-specific expression and it was previouslyshown that over-expression of same in the mouse forebrain enhancesrecognition memory and LTP [Wang H et al., Nat Neurosci (2004) 7:635-42].

In order to investigate whether miR-19 indeed regulates Adrb1 or ADCY1expression through its presumed target sequences on Adrb1-3′UTR orADCY1-3′UTR, an intact, or mutated forms of Adrb1-3′UTR (FIG. 5) orADCY1-3′UTR were cloned downstream of the luciferase gene in thePsicheck2 expression plasmid. In the mutated form of ADRb1-3′UTR all 4seed matches for miR-19b were absent (FIG. 5). In the mutated form ofthe partial ADCY1-3′UTR, only the conserved seed-match (out of 3) wasabsent.

Luciferase assay was used to determine the nature of interaction betweenmiR-19 and Adrb1-3′UTR and also between miR-19 and ADCY1-3′UTR. In HT22cells, that endogenously express low levels of miR-19, no difference wasfound between luciferase levels controlled by either intact or mutatedform of ADRb1-3′UTR (FIG. 6A). However, when miR-19b was over-expressedin HT22 cells, luciferase levels were significantly (approximately 2fold) lower when driven by the intact form relative to the mutated formof ADRb1-3′UTR (in addition to a general, seemingly non-specificreduction in normalized luciferase expression) (FIG. 6B). In HEK293Tcells that endogenously express high levels of miR-19b, luciferaseexpression levels regulated by ADRb1-3′UTR were 2-4 times lower thanthose expressed when regulated by the mutated form of ADRb1-3′UTR (FIG.6C).

MiRs knockdown (KD) system was used in order to manipulate miR-19 levelsin HEK293T cells. Namely, (1) miRCURY LNA KD probes (Exiqon, Mass., USAFIG. 6D), and (2) plasmid based knockdown sequence miArrest(Genecopoeia, Rockville, Md., USA, FIG. 6E). LNA-Anti-miR-19b enhancedluciferase levels expressed when regulated under ADRb1-3′UTR at about20% relative to control scrambled KD probe and had no effect on themutated form of ADRb1-3′UTR (FIG. 6D). Whereas, plasmid based miR-19bKD, caused up to 2 fold enhancement in luciferase expression regulatedby the intact form of ADRb1-3′UTR relative to Control KD sequence (FIG.6E). No full rescue of luciferase levels relative to that driven by themutant form of ADRb1-3′UTR was achieved. This may be explained either bymiR-19b specificity of the probe/genomic sequence (spearing miR-19aregulation), the high miR-19 levels in HEK293T cells that may bedifficult to fully down-regulate, or the effect of other possible miRNAsexpressed in HEK293T cells that may bind to the same seed-matchsequences on ADRb1-3′UTR.

Example 3A MiR-19a and MiR-19b are Upregulated in the PFC and AmygdalaFollowing Chronic Stress

Materials and Experimental Procedures

Animals and Housing

miR 17˜92 flx/flx Mice [Ventura A et al, Cell (2008) 875-86:(5)132; 7],are cross-bred with CamKIIa-Cre mice [Dragatsis I et al Genesis. (2000)26(2):133-5]. Transgenic Mice or Adult C57BL/6J male mice are housed ina temperature-controlled room (22±1° C.) on a reverse 12 hour light/darkcycle. Food and water available ad libitum. All experimental protocolswere approved by the Institutional Animal Care and Use Committee of TheWeizmann Institute of Science.

Generating Lentiviruses for miR-19b Manipulation in Adult Brain

MiR-19b KD sequence was cloned into a lentiviral plasmid following theRNA polymerase III-H1 promoter. In addition, Pre-miR-19b sequence wascloned following a neuronal specific promoter (Enhanced synapsin, ESyn)in a lentiviral plasmid. Lentiviruses are generated for both in-vivomiR-19b-KD and Pre-miR-19b-overexpression (OE) experiments. Theselentiviruses are used to manipulate miR-19b levels in target regionswhere miR-19 levels are found to be altered following abehavioral/pharmacological challenge.

Generating Mice Lacking miR-19 in the Forebrain

In order to generate mice lacking miR-19 in the forebrain, inventors arebreading mice carrying the gene encoding for Cre recombinase under theCamKIIa promoter, with mice carrying a conditional form of the miRscluster miR17-92. MiR-19 family includes miR-19a and miR-19b. In themouse genome miR-19b has two identical copies, miR-19b-1 and miR-19b-2.MiR19a and miR-19b-1 are located on the same miRNA cluster, namelymiR17-92, whereas miR-19b-2 is located at a different genomic locus,miR106a˜363. The latter seems to have little or no expression in mousetissues and therefore the knockout of miR17˜92 cluster is expected to beenough to enable a profound effect on miR-19a and miR-19b expressionlevels in the forebrain.

Behavioral/Pharmacological Challenges

Mice lacking miR-17˜92 cluster in the forebrain, or mice where miR-19was specifically manipulated (overexpressed or down-regulated (KD) inspecific brain regions) will be examined for expression levels of ADRb1,ADCY1 and other transcripts and gene products. These animals will bealso tested for anxiety like behavior, locomotor activity and memoryperformance. Furthermore, the levels of expression of miR-19a andmiR-19b are examined in different regions of interest (E.G thehippocampus, amygdala and forebrain) following an acute and chronicsystemic treatment with the Noradrenaline reuptake inhibitor Reboxetinein WT mice.

Results

The physiological link between miRNA-19 and Adrb1 was studied byassessing the level of miR-19a/b in the prefrontal cortex (PFC) of micethat were injected with Reboxetine, a noradrenalin reuptake inhibitor(NRI), either acutely or chronically (FIGS. 12A-12D). As shown in FIGS.12A-12D, miR-19 a/b levels were down regulated following acuteadministration of Reboxetine (FIG. 12A,12B) and upregulated followingchronic administration of Reboxetine (FIG. 12C,12D).

Next, the levels of miR-19 were assessed following stress by measuringthe levels of miR-19 a and b in the PFC and amygdale of mice subjectedto social defeat protocol (FIGS. 13A-13D). As shown in FIGS. 13A-13D,the levels of miR-19 a and b increased both in the PFC and amygdalafollowing chronic stress. These results illustrate the involvement ofmiR-19 in the regulation of the central stress response.

Example 3B miRNA-19 and Canabinoid Receptor 1 (CB1)

Materials and Experimental Procedures

Animals and Housing

As described in Example 3A, above.

Generating Lentiviruses for miRNA-19b Manipulation in Adult Brain

As described in Example 3A, above.

Results

CB1 is one of the most abundantly expressed GPCRs in the brain and isparticularly enriched in the cortex, amygdala, hippocampus, basalganglia, and cerebellum (FIGS. 15A-15B) [Herkenham M. et al., TheJournal of neuroscience: the official journal of the Society forNeuroscience (1991) 11:563-583; Mackie, K. Handbook of experimentalpharmacology (2005) 299-325]. CB1 receptors are highly expressed onaxons and axon terminals, where they are well positioned to modulateneurotransmission. Inventors found that CB1 contains 2 seed sites thatare compatible with miRNA-19.

A luciferase assay was used to determine the nature of interactionbetween miRNA-19 and CB1-3′UTR. When miRNA-19b was over-expressed inHT22 cells along with the 3′UTR of CB1, luciferase levels weresignificantly (50%) lower when compared to GFP over expressed with thesame 3′UTR (FIG. 14), supporting a possible role for miR-19 in theregulation of CB1 levels. Additional mutation experiments are performedto verify the role of the predicted miR-19 seed sequence to the observedregulation (as described for Adrb1 above).

Interestingly, previous studies have convincingly demonstrated that theconsolidation of aversive memories is facilitated by cross-talk betweenglucocorticoids, noradrenergic and cannabinoid signaling in thebasolateral nucleus of the amygdala (BLA) [Roozendaal, B. et al.Neurobiology of learning and memory (2006) 86:249-255]. A model proposedby Hill and McEwen [Hill M. N. and McEwen B. S. Proc of the Nat Acad ofSci of the USA (2009) 106:4579-4580] shows a possible mechanism ofaction in the BLA for memory consolidation (FIG. 16).

As shown in the present results, MiRNA-19 appears to regulate both Adrb1and CB1 in vitro. Over-expression and knockdown of miR-19 using e.g.lentiviruses delivered specifically to the BLA where it may alter thelevels of Adrb1 and CB1, are carried out as well as tests examining themice's performance in learning and memory paradigms such as fearconditioning with and without exposure to stressful challenges.

Example 3C Identification of Differentially Expressed miRNAs in MiceSubjected to Chronic Stress

Materials and Experimental Procedures

Immunoprecipitation of Ago2 Protein

Pools of 3 amygdalae from 3 animals that are part of the same group(“Susceptible”, “Resilient” or Control) were homogenized in NP40 bufferwhich was supplemented with RNase inhibitor, protease inhibitor andphosphates inhibitor. The samples were maintained on constant agitationfor 2 hours at 4° C. Samples were then centrifuged for 20 min at 12,000rpm at 4° C. in a micro centrifuge, the supernatant was placed in afresh tube kept on ice and the pellet was discarded. Magnetic protein Gbeads (Dynabeads, Invitrogen) were incubated with the Ago2 monoclonalantibody (WAKO) with rotation at room temperature for 10 minutes. Afterseveral washes the samples were added to the Ago2 coated protein G beadsand incubated over night at 4° C. under agitation. The following day thebeads were washed 3 times with PBS. For RNA purification the beads werehomogenized in RLT buffer (RNeasy kit, miRNA supplementary protocol).For western blot analysis the beads were boiled in sample buffer torelease the protein from the beads.

RNA Purification and Microarray

RNA from the Ago2 immunoprecipitation samples was isolated using theRNeasy plus kit (Qiagen) following Qiagen supplementary Protocol 1:Purification of total RNA containing miRNA. RNA for all other purposeswas isolated from frozen brain punches using miRNeasy mini kit (Qiagen)according to the manufacturer recommendation, and RNA integrity wasevaluated using the Agilent 2100 bioanalyzer. RNA derived from tissuesof stressed mice following Ago2 immunoprecipitation was further analyzedon Affymetrix miRNA 2.0 arrays (enriched RNA protocol) and AffymetrixMouse Gene 1.0 ST array.

Results

In order to identify and study differentially expressed miRNAs isolatedfrom the amygdala of mice subjected to chronic stress paradigm and/orassociated with “Resilient” or “Susceptible” behavioral phenotype, thesocial defeat protocol was used (see Methods section).

In order to identify a genuine connection between miRNAs and theirtarget gene's 3′ UTR following the social defeat paradigm, animmunoprecipitation (IP) of the Ago2 complex was performed and thepopulation of miRNAs and mRNAs co-precipitated was analyzed. When amature miRNA was formed it was incorporated to the RISC complex. Whilein the RISC complex, Ago2 facilitates the interaction between a specificmiRNA and its target mRNA 3′ UTR [Meister G. et al., Molecular cell(2004) 15:185-197] (FIG. 17A).

In order to verify that the Ago2 complex can indeed be precipitated withits bound RNA, the IP was performed on the amygdala of naive mice. TheIP was performed using protein G magnetic beads which were reacted withmonoclonal Ago2 antibody. As shown in FIG. 17B, a specific Ago2 band wasprecipitated from an extract of NIH 3T3 cells (FIG. 17B, lane 1) or froman extract of amygdala tissue (FIG. 17B, lane 2).

To demonstrate the specificity of the IP, a total brain sample wasdivided into two, where one was precipitated with anti Ago2 and theother with a control IgG1 nonspecific antibody. A specific Ago2 band waspresent only in the Ago2 precipitate (FIG. 17B, lanes 3, 4).

Therefore, by pulling down the Ago2 complex and analyzing the miRNA aswell as the mRNA populations in the precipitated material there was agreater chance to discover a correct connection between a given miRNAand its targeted mRNA 3′ UTR in specific brain regions.

Isolation of Ago 2 Associated RNA from Mice Amygdala Subjected to SocialDefeat Paradigm

Next, based on the specific results of the Ago2 IP experiment, the samestrategy was implemented in order to reveal potential differences inmiRNA and their target mRNAs in the brain of mice that were subjected tosocial defeat protocol.

After 10 days of the social defeat paradigm, mice were categorized into3 groups: Control, “Susceptible” and “Resilient”. A mouse wascharacterized as “Susceptible” when it exhibited social avoidance whenit encountered a new mouse from the same strain that attacked him duringthe social defeat paradigm. A mouse was characterized as “Resilient” ifit does not avoid the new aggressive mouse and interacts with it. Mostof the mice subjected to social defeat typically exhibit socialavoidance and therefore would be classified as “Susceptible”.Approximately only 10-20% of the mice in an experiment are expected tobe “Resilient”. Shown below is an example of the social avoidance testconducted.

As demonstrated in FIG. 18A, the mouse was placed alone in the socialmaze for 3 minutes for habituation. The camera tracked the mousemovements throughout the maze. In FIG. 18C, the same mouse was exposedto a novel ICR mouse that was placed beyond a divider. The cameratracked the mouse in the farthest corner of the arena distant from thelocation of the novel mouse. This response was considered as socialavoidance and therefore this mouse was classified as “Susceptible”. Incontrast, in FIG. 18B and FIG. 18D the mouse did not exhibit socialavoidance and therefore was classified as “Resilient”.

Forty mice underwent the social defeat paradigm and forty mice served ascontrol. Following the social avoidance test 9 “Resilient” mice, 9“Susceptible” mice and 12 control mice were selected for brainmicrodissection. Brain samples were collected 8 days after the socialavoidance test from the amygdala, BNST, PFC, dorsal raphe andhippocampus along with trunk blood.

Pools of 3 amygdala punches obtained from 3 different mice were combinedand the immunoprecipitation with anti Ago2 was performed. Following theIP, RNA was extracted from the precipitated material. After the pullingof 3 amygdalae from each group there were 3 RNA samples from the“Resilient” mice, 3 RNA samples from the “Susceptible” mice and 4 RNAsamples from the control mice—a total of 10 RNA samples. Each sample wastested in a mouse ST microarray as well as in miRNA array (bothAffymetrix). Genes and miRNAs that were up or down regulated in each ofthe 2 groups: “Susceptible” or “Resilient” relative to the controlgroup, were examined. If an interaction between a certain miRNA and atarget gene takes place inventors expected for an opposite correlationin their total levels. However, mRNA present in the RISC complex(precipitated with the anti Ago2) were expected to be in high levelsbecause they have not yet been fragmented, therefore while looking atthe array data inventors examined miRNAs and potential mRNA targets thatwere both either elevated or down regulated relative to the controlsample because this was an indication that they interacted in the RISCcomplex.

Microarray Results

Table 3, hereinbelow, illustrated the preliminary array results analyzedusing conventional filters.

TABLE 3A-B List of amygdalar miRNAs up regulated (Table 3A) or downregulated (Table 3B) following IP with Ago2. (Table 3A) Fold-ChangeFold-Change Upregulated Susceptible Resilient mmu-miR-301a_st 1.96 2.11mmu-miR-15a_st 1.66 1.87 mmu-miR-29a_st 1.42 1.82 mmu-miR-19b_st 1.972.34 mmu-miR-146b_st 1.55 1.94 mmu-miR-181d_st 1.54 1.64 mmu-miR-146a_st1.41 1.60 mmu-miR-27b_st 1.45 1.91 mmu-miR-20a_st 1.57 1.52mmu-miR-30a_st 1.34 1.65 mmu-miR-100_st 1.41 1.55 mmu-miR-153_st 1.441.92 mmu-miR-194_st 1.57 1.78 mmu-miR-30c_st 1.40 1.66 mmu-miR-23a_st1.51 1.70 mmu-miR-106a_st 1.62 1.61 mmu-miR-30b_st 1.43 1.70mmu-miR-195_st 1.59 1.98 mmu-miR-30e_st 1.36 1.56 mmu-miR-126-3p_st 1.581.76 mmu-let-7i_st 1.49 1.57 mmu-miR-434-5p_st 1.30 1.55 mmu-miR-376b_st1.64 1.99 mmu-miR-495_st 1.45 1.82 mmu-miR-369-5p_st 1.60 1.77mmu-miR-421_st 1.71 1.53 mmu-miR-543_st 1.52 1.69 mmu-miR-410_st 1.441.76 mmu-miR-34b-5p_st 2.18 1.53 (Table 3B) Fold- Fold-Change ChangeDownregulated Susceptible Resilient mmu-miR-210_st −1.59 −2.13mmu-miR-298_st −1.75 −2.08 mmu-miR-423-5p_st −1.68 −1.94 mmu-miR-346_st−1.74 −1.96 mmu-miR-139-3p_st −1.71 −2.13 mmu-miR-320_st −1.74 −2.03mmu-miR-485_st −1.53 −1.88 mmu-miR-491_st −1.53 −2.01 mmu-miR-31_st−1.30 −1.53 mmu-miR-92b_st −1.20 −1.53 mmu-miR-93_st −1.36 −1.50mmu-miR-125a-3p_st −1.32 −1.55 mmu-miR-134_st −1.47 −1.63mmu-miR-323-5p_st −1.43 −1.76 mmu-miR-345-5p_st −1.30 −1.62mmu-miR-341_st −1.36 −1.89 mmu-miR-370_st −1.33 −2.04 mmu-miR-433_st−1.49 −1.75 mmu-miR-455_st −1.40 −1.61 *For both Tables 3A-B, the datawas presented as fold change for “Susceptible” or “Resilient” micecompared with Control. Values in bold are significantly altered.

Several miRNAs, which have been significantly upregulated in the“Susceptible” and “Resilient” groups of mice, have been selected andillustrated in a heatmap (see FIGS. 19A-19B).

Gene Expression Array (mRNA)

TABLE 4 List of amygdalar mRNAs up regulated following IP with Ago2.Fold-Change Fold-Change Upregulated Susceptible Resilient Tnrc18 1.361.23 Ifi30 1.34 1.21 Adamts9 1.79 1.52 Fkbp5 1.35 1.26 Adh1 1.42 1.05Pxdn 1.32 1.19 Impdh2 1.41 1.02 Pdzd2 1.31 1.31 Csmd3 1.33 1.44 Usf11.33 1.20 A2m 1.71 1.09 Ccnd3 1.34 1.10 Rrh 1.33 1.02 Wfikkn2 1.40 1.07Fras1 1.48 1.34 Notch2 1.50 1.22 Fam38a 1.33 1.18 Hist1h3f 1.31 1.19Fam167a 1.31 1.05 Calml4 1.68 1.11 Tspan4 1.30 1.21 Dnahc6 1.38 1.07Jag2 1.31 1.19 Shank2 1.60 1.42 Dock6 1.33 1.10 Mamdc2 1.30 1.20 Sgms21.39 1.13 Iqub 1.51 1.11 Ubxn11 1.36 1.06 Wfdc2 1.53 1.11 Spef2 1.331.16 Fggy 1.31 1.14 Pcolce2 1.37 1.16 Thbs1 1.32 1.13 Dnahc7b 1.40 1.13Nt5dc2 1.41 1.12 Slc4a2 1.34 1.07 Adamts17 1.40 1.35 Plscr2 1.34 1.21Clic6 1.43 1.13 St6galnac2 1.38 1.08 Amigo2 1.33 1.06 Trio 1.33 1.15Lamb1-1 1.35 1.20 Sema3b 1.40 1.01 Fap 1.39 1.10 Frem1 1.51 1.20 Pon11.34 1.03 Plin4 1.43 1.24 Steap1 1.36 1.10 Rdh5 1.52 1.13 Cldn2 1.561.11 Frrs1 1.37 1.10 Spef2 1.36 1.07 Slco1a5 1.31 1.13 Ltc4s 1.35 1.17Mfsd7c 1.37 1.14 Acss3 1.32 1.16 Hif3a 1.36 1.17 Serpinb8 1.40 1.18Pcolce 1.36 1.16 Dnmt3a 1.20 1.19 GILZ 1.19 1.15 (Tsc22d3) Sdk2 1.291.36 Prg4 1.16 1.72 Fbn1 1.24 1.10 Slitrk6 1.11 1.28 Plxna1 1.30 1.16Plxnb2 1.25 1.10 Sema4b 1.29 1.14 *Data is presented as fold change for“Susceptible” or “Resilient” mice compared with Control. Values in boldare significantly altered.

TABLE 5 List of amygdalar mRNAs down regulated following IP with Ago2.Fold- Fold- Change Change Downregulated Susceptible Resilient Cyp2d10−1.22 −1.34 Lonrf1 −1.32 −1.31 Btn15 −1.64 −1.54 B2m −1.33 −1.20 Tekt5−1.36 −1.10 Prp2 −1.51 −1.02 Krtap5-1 −1.34 −1.10 Krtap5-4 −1.33 −1.10Klhl38 −1.38 −1.07 Th −1.42 −1.03 Pcsk9 −1.33 −1.20 Dnahc3 −1.39 −1.22Sgpp2 −1.37 −1.03 Opalin −1.49 −1.28

Several potential miRNAs and their putative targets in the brain areanalyzed.

Example 4A miR-15a and miR-15b as Regulators of the Stress Response

Materials and Experimental Procedures

Total RNA Extraction

Amygdala tissue was dissected 90 minutes following acute stressprocedure. Total RNA was isolated using miRNeasy kit (Qiagen) in orderto preserve miRNAs. Frozen brain punches were transferred into lysisbuffer and immediately homogenized. Neuronal primary cultures or N2acell cultures were lysed in-well, on ice. Further processing was doneaccording to the manufacturer's recommendation. RNA extracts were storedat −80° C. until use.

miRNA Array

miRNA differential expression was assayed by Agilent (Agilent, SantaClara, Calif., USA) or Affymetrix (Affymetrix, Santa Clara, Calif., USA)miRNA microarrays, according to the manufacturer's instructions. For theassessment of miRNA differential expression using the Agilent array, 100ng total RNA per sample (3 control samples and two acute stress samples)were each labeled and hybridized according to the manufacturer'sinstructions. Arrays were scanned using an Agilent microarray scanner.The data was extracted using the Agilent Feature Extraction software v9and analyzed using Partek® Genomics Suite (Partek Inc., St. Louis, Mo.,USA). Data from the GeneView.txt files were subject to logtransformation and quantile normalization. For the assessment of miRNAdifferential expression using the Affymetrix array, 1 μg total RNA persample (two control samples and two acute stress samples) were eachlabeled and hybridized according to the manufacturer's instructions.Arrays were scanned using an Affymetrix microarray scanner. The data wasextracted using the Affymetrix scanner software and normalized using thedefault parameters of the Affymetrix miRNAQCtool software (backgroundadjustment, quantile normalization, log transformation and thresholddetermination). The normalized data from the four files were importedinto Partek Genomics software. Genes not presented in any of themicroarrays were filtered out. Due to the difference in miRNAdistribution, different log ratio cutoffs (corresponding to about 1standard error for each array) were chosen for each array: 0.2 forAgilent and 0.4 for Affymetrix. miRNAs with log ratios greater than thecutoff were compared between arrays and the common miRNAs are reported.

Cloning of 3′ UTRs into Psicheck2 Luciferase Expression Plasmid

3′UTR sequence of CRFR1 was PCR amplified from mouse genomic DNA. 3′UTRPCR fragments were ligated into pGEM-T easy vector (Promega) accordingto the manufacturer's guidelines, and further subcloned into a singleNotI site at the 3′ end of luciferase in the Psicheck2 reporter plasmid(Promega). Cloning orientation was verified by diagnostic cuts and bysequencing.

Transfections and Luciferase Assay

HEK293T cells were grown on poly-L-lysine in 48-well format to a 70-85%confluence and transfected using Polyethyleneimine with the followingplasmids: Psicheck2-3′UTR plasmid, pre-mmu-miR-15 over-expression inpEGFP plasmid or pEGFP plasmid alone (clontech). 24 hours followingtransfection, cells were lysed and luciferase reporters activity wereassayed as previously described [Chen A. et al. Mol Endocrinol (2005)19: 441-58]. Renilla luciferase values were normalized to controlfirefly luciferase levels (transcribed from the same vector but notaffected by 3′UTR tested) and averaged across six well repetitions percondition.

Results

miR-15a and miR-15b emerged as up regulated 90 minutes following acuterestraint stress (FIG. 7A-7B). Both miR-15a and miR-15b werebioinformatically predicted to target CRFR1-3′UTR (FIG. 7C). In-Vitrooverexpression of miR-15b in HEK293T cells significantly reduced thelevels of luciferase expression controlled by CRFR1-3′UTR (FIG. 7D).

Example 4B The Effect of miR15 on FKBP5

Materials and Experimental Procedures

As illustrated in Example 4A, hereinabove.

Results

According to the array results, miR-15a and FK506 binding protein 5(also known as FKBP5) were both up regulated in the “Susceptible” and“Resilient” mice relative to the control group (FIGS. 20A-20B),suggesting their up regulation in the RISC complex as a result ofchronic stress.

Genetic studies have identified a role for FKBP5 in posttraumatic stressdisorder, depression and anxiety. For example, single nucleotidepolymorphisms (SNPs) in FKBP5 have been found to interact with childhoodtrauma to predict severity of adult posttraumatic stress disorder (PTSD)[Binder, E. B. et al., Nature genetics (2004) 36:1319-1325]. Thesefindings suggest that individuals with these SNPs who are abused aschildren are more susceptible to PTSD as adults. FKBP5 has also beenfound to be less expressed in individuals with current PTSD [Yehuda, R.et al., Biological psychiatry (2009) 66:708-711]. The FKBP5 gene hasbeen found to have multiple polyadenylation sites and is statisticallyassociated with a higher rate of depressive disorders [Binder et al.supra].

Further analysis of the 3′ UTR of FKBP5 revealed that it has oneconserved seed match sequence to miR-15 (FIG. 20C).

If indeed miR-15a regulates FKBP5 mRNA, it was expected that while bothmiR-15a and FKBP5 would be up regulated in the Ago-2 precipitate (asshown by the microarray results, FIG. 20B), the total levels of eithermRNA or protein of FKBP5 in the amygdala sample would be decreased.

In order to examine whether the interaction between miR-15a and FKBP5takes place in the amygdale, a real time PCR analysis on total RNAsample obtained from the amygdala of “Susceptible” and control mice wasperformed. As shown in FIGS. 21A-21B, miR-15a levels were increased intotal RNA extract taken from susceptible mice whereas FKBP5 levels weredecreased. These results indicated that miR-15a represses FKBP5 levelsin the amygdala following chronic stress condition.

Cloning the intact and mutated 3′ UTR forms of FKBP5 for luciferaseassay analysis are performed in order to find whether a directinteraction between miR-15a and FKBP5 occurs in vitro.

In addition to FKBP5, miR-15 can potentially regulate a number of genesthat are involved in the stress response including Stx1a (syntaxin 1a),Sgk1 (serum/glucocorticoid regulated kinase) and Adrb2 (FIG. 22).

Example 4C miR-181 Regulates Glutamate Receptors

Materials and Experimental Procedures

Cloning of 3′ UTRs into Psicheck2 Luciferase Expression Plasmid

3′UTR sequences of Grm1, Grik3, Grm5,Grik2 and Grm7 were PCR amplifiedfrom mouse genomic DNA. 3′UTR PCR fragments were ligated into eitherpGEM-T easy vector (Promega) or pJET1.2 vector (Fermentas) according tothe manufacturer's guidelines, and further subcloned into a single NotIor XhoI site at the 3′ end of luciferase in the Psicheck2 reporterplasmid (Promega). Cloning orientation was verified by diagnostic cutsand by sequencing.

Chronic Social Defeat

Mice were subjected to a social defeat protocol as previously described[Krishnan V. et al. Cell (2007) 131: 391-404]. Briefly, the mice wereplaced in a home cage of an aggressive ICR mouse where they physicallyinteracted for five minutes. During this time, the ICR mouse attackedthe intruder mouse and the intruder displayed subordinate posturing.Perforated clear plexiglass dividers were then placed between theanimals and the mice remained in the same cage for 24 hours to allowsensory contact. The procedure was then repeated with an unfamiliar ICRmouse for each of the next 10 days.

Results

miR-181d levels were significantly increased in mice suffering fromchronic stress (FIG. 23). In an attempt to find interactions betweenmiR-181 and potential mRNA targets, Inventors discovered that miR-181can potentially regulate many types of glutamate receptors. In general,glutamate receptors can be divided into two groups, lonotropic glutamatereceptors (iGluRs), which form the ion channel pore that activates whenglutamate binds to the receptor, and Metabotropic glutamate receptors(mGluRs), which indirectly activate ion channels on the plasma membranethrough a signaling cascade that involves G proteins.

Of the many specific subtypes of glutamate receptors, it is customary torefer to primary subtypes by a chemical which binds to it moreselectively than glutamate. The research, though, is ongoing, assubtypes are identified and chemical affinities measured. Severalcompounds are routinely used in glutamate receptor research andassociated with receptor subtypes:

TABLE 6 Glutamate receptors categorized into subgroups Name Type NMDAreceptor Ionotropic Kainate receptor AMPA receptor mGluR Metabotropic

As illustrated in FIGS. 24 and 25, out of all the conserved predictedtargets of miR-181, there are 6 glutamate receptors (Grm1, Grik3, Grm5,Gria2, Grik2 and Grm7).

It has been shown previously that miR-181a controls Gria2 surfaceexpression in hippocampal neurons [Saba. R. et al., Molecular andCellular Biology (2012) 32(3):619-32]. Luciferase assays are beingperformed in order to verify the miRNA-mRNA interaction. Furthermore, aconditional miR-181 KO mice line are crossed with a specific cre linethereby obtaining a deletion of miR-181 in specific brain nuclei.

Example 5A MiR-182 a Fine Tuner of Normal Neuronal Activity and ofPsychopathological Behavior

Materials and Experimental Procedures

Cloning of 3′ UTRs into Psicheck2 Luciferase Expression Plasmid

3′UTR sequence of Htr1a was PCR amplified from mouse genomic DNA. 3′UTRPCR fragments were ligated into pGEM-T easy vector (Promega) accordingto the manufacturer's guidelines, and further subcloned into a singleNotI site at the 3′ end of luciferase in the Psicheck2 reporter plasmid(Promega). Cloning orientation was verified by diagnostic cuts and bysequencing.

Transfections and Luciferase Assay

HEK293T cells were grown on poly-L-lysine in 48-well format to a 70-85%confluence and transfected using Polyethyleneimine with the followingplasmids: Psicheck2-3′UTR plasmid, pre-mmu-miR-182 over-expression inpEGFP plasmid or pEGFP plasmid alone (clontech). 24 hours followingtransfection cells were lysed and luciferase reporters activity wereassayed as previously described [Chen A. et al. Mol Endocrinol (2005)19: 441-58]. Renilla luciferase values were normalized to controlfirefly luciferase levels (transcribed from the same vector but notaffected by 3′UTR tested) and averaged across six well repetitions percondition.

Chronic Social Defeat

Mice were subjected to a social defeat protocol as previously described[Krishnan V. et al. Cell (2007) 131: 391-404]. Briefly, the mice wereplaced in a home cage of an aggressive ICR mouse and they physicallyinteracted for five minutes. During this time, the ICR mouse attackedthe intruder mouse and the intruder displayed subordinate posturing.Perforated clear plexiglass dividers were then placed between theanimals and the mice remained in the same cage for 24 hours to allowsensory contact. The procedure was then repeated with an unfamiliar ICRmouse for each of the next 10 days.

Microdissection of the Raphe Nucleus and Plasma Collections

Brain samples were taken from mice raphe nucleus (RN) after removing thebrain and placing it on acryl brain matrix (Stoelting). Slices weretaken using standard razor blades (GEM) based on designated anatomicalmarkers. Blunted 14G syringes were used to extract the RN region from 3mm slices removed from the matrix.

microRNA Purification and Quantitative RT-PCR Expression Analysis

mRNAs, including microRNAs, were isolated from sorted neurons, frozenbrain punches and plasma using miRNeasy mini kit (Qiagen) according tothe manufacturer instructions, and treated using miScript Reversetranscription kit miRNA to generate cDNA. cDNA samples were thenanalyzed using SYBR®Green PCR kit (Qiagen) according to themanufacturer's guidelines in AB 7500 thermocycler (Applied Biosystems).Specific primers for each miR were used together with the commercialuniversal primer, while U6 snRNA was used as internal control.

Cloning of miR182 Over Expression Viral Vector

Pre-miR-182 was amplified by PCR from mouse genomic DNA with primersadding restriction enzyme AgeI sites and then was inSlc6a4ed to pGEM-TEasy vector (Promega, Madison, Wis.). After sequencing of pGEM-T Easyand digestion of both pGEM-T Easy and pEGFP vector (Clontechlaboratories Inc., Mountain View, Calif.) with the AgeI, the prematuremiR-182 sequence was ligated to the pEGFP vector to construct theexpression plasmid pEGFP-miR-182. Afterwards, pEGFP-miR-182 was cut byBamHI and BsrGI in parallel to cutting pCSC-E/Syn-eGFP plasmid with thesame enzymes, and the miR-182-eGFP sequence was ligated to pCSC-E/Syn toconstruct pCSC-eSNY-pre-miR-182-eGFP plasmid which was confirmed byrestriction endonuclease analysis and DNA sequencing.

Production of Lentiviral Vectors

Recombinant lentiviruses were produced by transient transfection inHEK293T cells, as previously described [Naldini L et al., Proc Natl AcadSci USA (1996) 93:11382-8]. Briefly, infectious lentiviruses wereharvested at 48 and 72 hours post-transfection, filtered through 0.45μm-pore cellulose acetate filters and concentrated byultracentrifugation.

Results

To date miR-182 was reported mainly in cancer related studies such as anhuman lung adenocarcinoma cells, glioma, breast cancer, bladder cancer,melanoma and DNA repair. Additionally, miR-182 was found to be involvedin developmental processes such as inner ear and retinal development,and in the immune system in activation of T lymphocytes, and in lupusdisease. In the nerves system mi-R182 was implied in sensoryorgan-specific rat dorsal root ganglia, and as a circadian clockmodulator, while a correlation between genetic variants of pre-miR-182were found in major depression patients [Saus E et al., Hum Mol Genet.(2010) 19(20):4017-25]. Additionally, miR-182 was listed among other 12miRs as down-regulated in resilient to learned helpless behaviors malerats prefrontal cortex [Smalheiser N R et al., Int JNeuropsychopharmacol. (2011) 1-11].

Bioinformatical analysis of Htr1a 3′UTR performed as part of the 5HTmiRs microarray analysis implied a possible targeting of this gene bymiR-182. Therefore, inventors performed in vitro testing via aluciferase assay, which revealed a strong repression of Ht1a 3′UTR bymiR-182 (FIG. 8). Two conserved seed matches sequence for miR-182appeared in Htr1a mouse 3′ UTR.

Regulation studies indicated a strong tendency of down-regulation ofmiR-182 expression levels in the RN of adult male mice exposed tochronic social defeat compared to controls (FIG. 9) suggestinginvolvement of miR-182 in the molecular response to environmentalstimulus known to induced depression-like behaviors.

Further bioinformatics analysis generating targeting predictions formiR-182 in two databases revealed a long list of potential targets,including genes related to neuronal activity both in normal and inpathological conditions (FIG. 10).

In order to further test miR-182 in vitro for identification of specificmiR target interactions, and to reveal miR-182 role in regulation ofnormal and pathological behaviors in vivo, plasmid and lentiviralsystems for manipulation of miR-182 were developed. Neuronal specificover-expression lentiviruses were manufactures (FIG. 11A) and tested invitro in the neuronal cell line N2a. These results demonstratedincreased miR-182 levels in cells infected with miR-182 over-expressionlentiviruses compared to control (FIG. 11B). Knockdown plasmid sequencespecific for miR-182 named miArrest (Genecopoeia, Rockville, Md., USA,FIG. 11C) was purchased and sub-cloned to viral constructs (FIG. 11C).These systems are tested in cell culture and by site specific injectionto adult mice brains.

Null mice for miR-182 are developed in order to investigate the miRsrole in retina development. Recently, inventors obtained breading pairsfor this line, and upon a generation a colony, miR-182 KO and their WTliter mates are being phenotyped behaviorally and physiologically.

Example 5B Regulation of miR182 Expression Levels by Acute Stress

Materials and Experimental Procedures

As Described in Example 5A, Hereinabove

Results

The effect of acute stress on miR182 level was examined. As illustratedin FIG. 26, acute immobilization stress led to decreased miR182expression levels in mice raphe nucleus (RN) 24 hours followinginduction of stress (P<0.01). miR182 demonstrated reduced expressionlevels in the raphe nucleus both following acute and chronic stress,suggesting it has a role in modulation the molecular responses to stressin the raphe nucleus, possibly by effecting its target gene Ht1amodulating 5-HT levels in the synapse.

miR-Target Interaction Assay for miR182 Predicted Target Genes

Using a luciferase assay, eleven predicted target genes of miR182,chosen after extensive bioinformatics, were examined (FIG. 27A). 3′UTRsof the target genes were tested in vitro to check if miR182 has arepresoric effect as measured by the activity of the conjugated reportergene luciferase. Out of the eleven genes tested three genes: Dscam (DownSyndrome Cell Adhesion Molecule), L1cam (Cell adhesion molecule L1) andTsnax (Translin-associated protein X) had demonstrated represoric effectby miR182 as in luciferase assay (FIG. 27A). When testing the 3′UTR ofthe listed above target gene of miR182 a conserved seed match sequencefor miR182 was observed both in Tsnax, L1cam and Dscam, suggesting thismiR-target interaction had a functional role (data not shown).

Next, the direct represoric effect of miR182 on these three genes wasverified. Therefore, the 3′UTRs was mutated to remove miR182 seed matchsequence and compared the regular 3′UTRs to the mutated one in vitro byluciferase assays. miR182 represoric effect on L1cam 3′UTR was abolishedwhen mutated its seed match sequence (FIG. 27B), and similarly theeffect of miR182 on Tsnax was abolished in the mutated 3′UTR (FIG. 27C)indicating miR182 targeted this gene directly. Similar verification forDscam and Htr1a with mutated 3′UTR are performed.

A mice model lacking miR182 is used to study the interaction betweenmiR182 and its target genes in vivo. Inventors are examining thebehavioral phenotype of miR182KO mice in tests for social behavior,learning and memory, and schizophrenia-like behaviors.

Example 6 Regulation of miR135 Levels in the Plasma and Brain of AdultMice

Materials and Experimental Procedures

Cloning of miR135 Overexpression Viral Vectors and of miR135 KD ViralVector

miR135b KD plasmid pEZX-H1-miR135KD-CMV-mCherry and controlpEZX-H1-control KD-CMV-mCherry were purchased from GeneCopeia (USA). H1promoter and the KD sequence were amplified using primers with flankingNheI site and ligated to pGEM-T Easy. After sequencing of pGEM-T Easyand digestion of both pGEM-T Easy and p156-pRRL-CMV-GFP with the NheIsite, H1-KD miR and nicked p156 were ligated to generatep156-pRRL-H1-miR135bKD-CMV-GFP and p156-pRRL-H1-control KD-CMV-GFP.

Behavioral Assessments

All behavioral assessments were performed during the dark phasefollowing habituation to the test room for 2 hours before each test.

Light/Dark Transfer Test

The light/dark transfer test apparatus and experimental conditions wereas previously described. Briefly, the apparatus contained 2 chambers, adark covered one, in which the mice were placed in the beginning of thetest, and brightly lighted chamber, to which it can transfer freelyduring the 5 minutes test. Time spent in the light compartment, distancetraveled in light, latency to visit the light chamber and number oflight-dark transitions were quantified with a video tracking system(VideoMot2; TSE Systems, Bad Hamburg, Germany).

Open-Field Test

The open field test was performed in a 50×50×22 cm white box, lighted in120 lux, in which the mice were put for a 10 minutes test. Time spent inthe center, number of visits in the center, latency to visit the center,number of rearing and total distance traveled were quantified using avideo tracking system (VideoMot2; TSE Systems, Bad Hamburg, Germany).

Elevated Plus Maze Test

This test apparatus had a plus shape and contained 2 barrier walls and 2open very low lighted (6 lux) arms. The number of entries, distancetraveled and the time spent in the open arms was automatically scoredusing video tracking system (VideoMot2; TSE Systems, Bad Hamburg,Germany) during the 5 minutes test.

Results

The effects of antidepressant administration on miR135 levels weretested in brain sites known to be innervated by serotonergic neuronsfrom the RN and involved in mood regulation, the amygdala (AMY) and theprefrontal cortex (PFC). In the AMY both miR135 variants wereupregulated by acute serotonin reuptake inhibitors (SSRI) and thenoradrenaline reuptake inhibitors (NRI) but not by chronicadministration of these drugs (P=0.0001 for SSRI, p=0.003 for NRI formiR135a, FIG. 28A; p=0.0001 for SSRI and p=0.003 for NRI for miR-135b,FIG. 28B). At the PFC, miR135b levels were upregulated by acute SSRI andNRI (P=0.0183 for SSRI and 0.0013 for NRI FIG. 28C) but miR135a levelswere not significantly altered (FIG. 28D). Additionally, chronic SSRIled to decreased miR135a and miR135b levels in the PFC (P=0.0241 formiR135a FIG. 28C, and P=0.0067 for miR135b FIG. 28D).

Additionally, miR135 levels in the circulation were tested following thesocial defeat paradigm. miR135a (P=0.0089; FIG. 29A) and miR135b(P=0.0033; FIG. 29B) levels were increased in the plasma of mice exposedto chronic social defeat compared to control mice as measured in realtime PCR. Thus, the present results demonstrated that miR135 in plasmawas upregulated following chronic stress, known to inducedepression-like behaviors in mice, and robustly decreased byantidepressant administration. These finding suggest miR135 levels inthe plasma as a biomarker for serotonergic-related depressive states.

Example 7 Establishment of miR135 Knockdown System; Cloning,Lentiviruses Generation and In Vitro and In Vivo Validations

Materials and Experimental Procedures

Cloning of miR135 KD Viral Vector

miR135b KD plasmid pEZX-H1-miR135KD-CMV-mCherry and controlpEZX-H1-control KD-CMV-mCherry were purchased from GeneCopeia (USA). H1promoter and the KD sequence were amplified using primers with flankingNheI site and ligated to pGEM-T Easy. After sequencing of pGEM-T Easyand digestion of both pGEM-T Easy and p156-pRRL-CMV-GFP with the NheIsite, H1-KD miR and nicked p156 were ligated to generatep156-pRRL-H1-miR135bKD-CMV-GFP and p156-pRRL-H1-control KD-CMV-GFP.

Results

To evaluate the effect of decreased miR135 levels in RN on mice5-HT-related behaviors, a plasmid based miR135b inhibitor was utilizedand its efficiently was tested in a luciferase assay. In this assay,HEK293T cells were co-transected with miR135OE, miR135KD and 3′UTRplasmids, and the ability of miR135bKD plasmid to block the repressingeffect of miR135 on Slc6a4 and Htr1a 3′ UTR was tested. miR135brepresoric effect of Htr1a 3′UTR was blocked by miR135KD plasmid (FIG.30A). Similarly, miR135b effect on Slac6a4 3′UTR was blocked by miR135KD(FIG. 30B). These results indicate that miR135KD plasmid indeed blocksthe biological activity of miR135.

miR135KD sequence and a control sequence were sub-cloned to a viralvector (FIG. 30C) and lentiviruses expressing the different knockdown(KD) sequence were generated. In order to test the lentiviruses' abilityto infect brain tissue, mice RN were infected with either one of thelentiviruses. Indeed, infection caused expression of GFP (FIGS. 30D-30E)demonstrating the ability of miR135bKD lentiviruses to infect braintissue.

Example 8 Behavioral Effects of miR135 Knock Down in Adult Mice RN

Materials and Experimental Procedures

Behavioral Assessments

mice were behaviorally characterized by using tests for anxiety anddepression-like behaviors as described in Example 6 above.

Results

Following the in vitro and in vivo validation of miR135KD lentiviruses,they were used to manipulate miR135 levels in the RN and to test theireffect on mice behavior. Adult mice were injected either with miR135KDlentiviruses, or KD control lentiviruses to RN and following recoveryperiod were tested for anxiety and depression-like behaviors. SincemiR135 represses negative regulators of 5-HT, we expected miR135KD tolead to decrease 5-HT levels in the synapse and by that to increasedanxiety and depression-like behaviors.

In the open field test no differences were observed between the groups(FIG. 31A), however in the elevated pulse maze test, miR135KD micedemonstrated higher anxiety-like behavior by demonstrating a tendency tospend less time in the open arms (P=0.0644) and to visit less times inthe open arms (P=0.0572 FIG. 31B). Additionally miR135KD mice walkedsignificantly less distance in the open arms (P=0.0433) and had a longertendency to visit in open arms (P=0.0124 FIG. 31B). Similarly, in thedark light test performed under basal stress conditions, miR135KD micedemonstrated a significant increased anxiety-like behavior compared tothe controls by spending less time in the light (P=0.0454 FIG. 31C),visiting less times in the light chamber (P=0.0107 FIG. 31D) and walkinga smaller distance in the light chamber (P=0.0402s FIG. 31E). Theresults illustrated a decrease in miR135 levels 40 min and 24 hoursafter acute stress (FIG. 30A-30B), therefore, the present theory wasthat stressed miR135KD mice would not differ from their controls inanxiety-like behaviors when tested following acute stress, since thecontrol mice would also have a decreased miR135 levels due to thestress. Indeed, there was no difference between the groups whenre-tested in the dark light transfer test in any of the parameters, bothwhen tested 40 minutes or 24 hours after acute stress (FIG. 31C-31E).

Depression-like behaviors of miR135KD were tested both under basalconditions and following pharmacological manipulation. Since miR135levels were showed to increase in the RN following SSRI administration(FIG. 31E), the speculation was that the reduction of miR135 levels maylead to reduced response to SSRI. In the tail suspension test performedboth in basal levels and after SSRI administration, there was nodifference between miR135b KD mice and control KD mice in immobilitytime (FIG. 31F), and the expected decrease in immobility time due toSSRI treatment was observed (P<0.0008). However, in the forced swimtest, additionally to the main effect for SSRI injection (P<0.0001),miR135KD mice injected with SSRI were more immobile in the last 2minutes of the test compared to control KD mice (P=0.0141 5 minute,P=0.0404 6 minute; FIG. 31G suggesting attenuation of SSRIantidepressant effects by reducing miR135 levels in the RN. This resultimplies that miR135 is part of the endogenous alternation leading tobehavioral changes caused by SSRI.

Example 9 miR135 Overexpression in 5-HT Neurons

Materials and Experimental Procedures

Mice Over Expressing miR135a in 5-HT Neurons were Compared to theirlittermates controls both in expression levels of miR135 and its targetgenes and behaviorally.

Results

The effects of manipulating miR135 levels specifically in 5-HT neuronsin the RN of mice was tested for anxiety and depression-like behaviors.For that purpose, a genetic system was developed using the Cre-loxPsystem. Specifically, the 5-HT specificity was obtained using the ePetCre mice expressing Cre recombinase specifically in the 5-HT RN positiveneurons and miR135 overexpression was performed by crossing the5-HT-specific Cre line (ePet Cre) with transgenic mouse line withconditional overexpression for miR135a (FIG. 32).

miR135 expression level in the RN of mice overexpressing miR135specifically in 5-HT neurons (miR135OE) was tested by real time PCR formiR135 and compared to control mice, positive for the miR135 conditionaloverexpression allele but negative for ePet CRE. miR135OE micedemonstrated near to 2 fold overexpression compared to control mice(FIG. 33A; P<0.05). Overexpression levels of miR135 were similar tolevels measured in the RN of mice following SSRI administration,suggesting this mice line was a good model for studying miR135antidepressant characteristics. Additionally, miR135 target gene mRNA,Slc6a4 (FIG. 33B; P<0.05) and Htr1a (FIG. 33C; P<0.1) weredown-regulated in the RN of miR135OE mice compared to controldemonstrating in vivo repression by miR135 of its target genes.

In order to test miR135 overexpression specifically in 5-HT neurons,miR135OE mice and their littermates controls were exposed to chronicsocial defeat paradigm, a procedure know to induce depression andanxiety-like behaviors, and subsequently were tested for anxiety anddepression-like behaviors.

miR135OE mice demonstrated increased anxiety-like behaviors followingsocial defeat compared to control liter mates. In the open field, atendency for increased anxiety was observed in miR135OE mice time andvisit number to the center (P<0.1, FIG. 34A). While in the dark lighttransfer test miR 135OE mice spent more time in light (P<0.05, FIG. 34B)and spent less time in the light chamber (P<0.01, FIG. 34B). Similarresults were observed in the elevated pulse maze (P<0.05, FIG. 34B)while miR135OE mice spent more time in the open arms (P<0.05, FIG. 34C)and traveled larger distance in the open arms (P<0.05, FIG. 34C).

Depression-like behaviors of miR135OE mice following social defeat werelower than of the control litter mates. A tendency towards decreasedimmobility time of the miR135OE mice compared to controls was observedin the tail suspension test (P<0.1, FIG. 34D), along with a significantdecreased immobility time in the forces swim test (P<0.05, FIG. 34E).

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by into thespecification, to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference. In addition, citation oridentification of any reference in this application shall not beconstrued as an admission that such reference is available as prior artto the present invention. To the extent that section headings are used,they should not be construed as necessarily limiting. In addition, anypriority document(s) of this application is/are hereby incorporatedherein by reference in its/their entirety.

What is claimed is:
 1. A method of treating a medical condition in whicha low glutamate receptor level is therapeutically beneficial in asubject in need thereof, the method comprising administering to saidsubject miR-181 or a precursor thereof or expressing in a cell of saidsubject an exogenous polynucleotide encoding a miR-181 or a precursorthereof, thereby treating the medical condition.
 2. The method of claim1, wherein said medical condition is selected from the group consistingof a seizure, a Huntington's disease, a schizophrenia, a fragile Xsyndrome, a generalized anxiety disorder, a memory impairment and acancer.
 3. The method of claim 1, wherein said subject is a humansubject.
 4. The method of claim 1, wherein said miR-181 is as set forthin SEQ ID NO: 85-94.
 5. The method of claim 1, wherein said miR-181comprises a modification selected from the group consisting of amodified backbone, a modified internucleoside linkage and a modifiedbase.
 6. The method of claim 5, wherein said modification is selectedfrom the group consisting of a phosphorothioate, a chiralphosphorothioate, a phosphorodithioate, a phosphotriester, an aminoalkylphosphotriester, a methyl phosphonate, an alkyl phosphonate, a chiralphosphonate, a phosphinate, a phosphoramidate, anaminoalkylphosphoramidate, a thionophosphoramidate, athionoalkylphosphonate, a thionoalkylphosphotriester, a boranophosphate,a peptide nucleic acid (PNA), a 2′-O-methoxyethyl, a 2′-O-methyl and a2′-fluoro.
 7. The method of claim 1, wherein said miR-181 comprises amodification in both a sugar and an internucleoside linkage.
 8. Themethod of claim 1, wherein said cell is a neuroglia cell.
 9. An isolatedcell comprising a nucleic acid construct expressing a miR-181 or aprecursor thereof under a transcriptional control of a cis actingregulatory element.
 10. The isolated cell of claim 9, wherein said cellis a neuroglia cell.
 11. The isolated cell of claim 9, wherein saidmiR-181 is as set forth in SEQ ID NO: 85-94.
 12. A nucleic acidconstruct comprising a nucleic acid sequence encoding a miR-181 or aprecursor thereof, said nucleic acid sequence being under atranscriptional control of a cis acting regulatory element.
 13. Thenucleic acid construct of claim 12, wherein said cis acting regulatoryelement is active in a neuroglia cell or a cardiac cell.
 14. Apharmaceutical composition comprising the nucleic acid construct ofclaim 12 and a pharmaceutically acceptable carrier or diluent.
 15. Amethod of regulating an expression of a glutamate receptor gene in aneuroglia cell, the method comprising modulating an activity orexpression of miR-181 or a precursor thereof in said neuroglia cell,thereby regulating the expression of the glutamate receptor gene. 16.The method of claim 15, wherein said glutamate receptor gene is selectedfrom the group consisting of glutamate receptor metabotropic 1 (Grm1),glutamate receptor ionotropic kainate 3 (Grik3), glutamate receptormetabotropic 5 (Grm5), glutamate receptor ionotropic kainate 2 (Grik2)and glutamate receptor metabotropic 7 (Grm7).